Sprayable aqueous composition comprising glyceryl trinitrate

ABSTRACT

The present application relates to a pharmaceutical preparation comprising (a) from 0.15 to 3 weight percent of glyceryl trinitrate, (b) from 40 to 95 weight percent water, and (c1) from 2 to 10 weight percent of at least one water soluble polymer, or (c2) from 1 to 10 weight percent of at least one water soluble polymer and 5 to 20 weight percent of ethanol.

This is the national phase under 35 U.S.C. § 371 of InternationalApplication No. PCT/EP2014/076020, filed on Nov. 28, 2014, which claimspriority to and the benefit of EP Application No. 13005562.7, filed onNov. 29, 2013, the entire contents of each of which are incorporatedherein by reference.

The invention concerns pharmaceutical preparations with the activesubstance glyceryl trinitrate in the form of an aqueous solutioncomprising a water soluble polymer, the use of the pharmaceuticalpreparation, a process for the preparation of the pharmaceuticalpreparation and a kit comprising the pharmaceutical preparation.

Glyceryl trinitrate (nitroglycerin, abbr. GTN) is a pharmaceuticalactive substance that is used among others for treating angina pectorisattacks. It is especially useful in emergency situations, when thepharmaceutical form must guarantee a quick onset of action. Sublingualsprays have proven highly efficacious because spraying the formulationinto the mouth represents a direct and quick procedure to apply the doseonto a great part of the mucosa. GTN is quickly absorbed from the mucosaand can act within seconds.

GTN containing sprays may be formulated with or without the addition ofa propellant. Propellant sprays are disclosed for example in the U.S.Pat. No. 3,155,574, in the European patent application EP 0 461 505 andthe German application DE 32 460 81. A GTN containing formulationwithout propellant for use as a pump spray is described in the Europeanpatent application EP 0 448 961.

GTN is a medium polarity liquid with limited water solubility, but itdissolves easily in a plurality of other solvents. Spray formulationsmay also be based on non-aqueous lipophilic, on non-aqueous solventsthat are miscible with water or on solvent systems containing water. Forexample, the patent EP 0 448 961 describes a rather lipophilicpreparation with a preferred content of triglycerides of 80% and ethanolas a co-solvent in a concentration of 20%. The propellant sprayaccording to the teaching of patent application EP 0 927 032 thatcontains approximately 30% triglycerides beside the propellant can alsobe characterized as highly lipophilic. In contrast, the propellant sprayaccording to U.S. Pat. No. 3,155,574 with 25% ethanol belongs to thesecond group of formulations. Also DE 3 922 650 A1 discloses apropellant free spray formulation that contains no water: GTN isdissolved in a plurality of polar solvents. In the European patentapplication EP 0 471 161 a pump spray is described that containsethanol, 1,2-propandiol and 42% water as solvents. The concentration ofmarketed GTN sprays is usually below 1%, because the therapeuticallyeffective dose for treating an angina pectoris attack lies below 1 mg.

The solubility of nitroglycerin in water is about 1 mg/ml. In theEuropean patent EP 0 108 248B1 solutions of GTN in a concentration rangeof 0.08 to 0.11 weight percent are proposed for infusion. This lowconcentration is sufficient for a solution for intravenous infusion toachieve therapeutic effects, because a comparatively large volume of thesolution can be applied. For a topical pharmaceutical form however, thisis not true. When a volume of more than 100 μl is applied sublingually,there is a remarkable risk of swallowing the drug. The active substanceis resorbed from the gastrointestinal tract much more slowly thanthrough the oral mucosa and the blood concentration is further decreasedby a prominent first-pass effect. Therefore the necessary plasma levelsfor a rapid and effective treatment in an emergency situation can not bereached in this way. In a similar manner there is also an upper limitfor dermal application of a spray solution, because when larger amounts,e.g. more than 100 μl, are applied they may get lost by dripping orrunning away. To reach efficacious plasma levels the concentration ofthe active substance in a topical GTN containing solution needs to behigher than 1 mg/ml, for example not less than 1.5 mg/ml.

U.S. Pat. No. 5,698,589 describes a topical cream with a GTNconcentration between 0.2 and 1.5 weight percent. The active substanceis formulated in an emulsion. This galenic form is not appropriate forsublingual or oral application because of difficulties of dosing andpatient compliance. In general, such preparations are not sprayablebecause of their high viscosity. Consequently, they can not be sprayedonto the skin or into the oral cavity. The patient would have to removeit from its container, put it for example onto a finger and then applyit onto the skin or the oral mucosa. Therefore a topical cream is notappropriate. Moreover, emulsions contain emulsifiers and otherexcipients for topical application that may not be acceptable for oralpreparations because of their bad taste or toxicological properties.Finally the active substance needs to be released from the emulsion,before it can be absorbed. This release is hindered because of thecontent of oily or greasy components in the oil phase of the emulsionand thickeners in the water phase. This aspect is also a severe drawbackfor the dermal application.

Taken together a considerable need for sprayable GTN preparationsremains. They should be well tolerated after topical application andguarantee a sufficient uptake of the active substance into the body. Thepreparations should be easy to manufacture and to apply.

The object of the present invention is to deliver a stablepharmaceutical preparation in the form of an aqueous solution containingnot less than 0.15 weight percent of GTN.

This object is solved by a pharmaceutical preparation comprising:

-   (a) from 0.15 to 3 weight percent of glyceryl trinitrate,-   (b) from 40 to 95 weight percent water, and-   (c1) from 2 to 10 weight percent of at least one water soluble    polymer,    or-   (c2) from 1 to 10 weight percent of at least one water soluble    polymer and 5 to 20 weight percent of ethanol.

According to the present invention, the term “weight percent” alwaysrefers to the weight of the pharmaceutical preparation. In other words,a pharmaceutical preparation according to the invention always comprises100 weight percent.

In the context of the present invention, it has been surprisingly foundthat by the use of water soluble polymers the active substance GTN staysin solution without an intolerable increase of viscosity of the solutionwhich would e.g. compromise sprayability of a sprayable embodiment ofthe invention. Therefore, such a pharmaceutical preparationcharacterized by low viscosity as defined herein can be prepared when,apart from usual excipients, a least one water soluble polymer ispresent that ameliorates the well-documented and typical insolubility ofthe active substance, GTN. This is illustrated by representativeGTN-containing preparations described in Examples 1 to 4 herein. Thefindings documented herein stand contrary to approaches heretoforeutilized by formulation chemists working to prepare improved,clinically-useful GTN-containing liquids. First, the water content ofthe present invention is substantially above that currently understoodto be possible when working with GTN. Second, the combination of highwater content and a water-soluble polymer resulting in a clinically- andcommercially-useful GTN-containing aqueous formulation is unexpected inview of the state of the art. And, third, the viscous properties of thepresent invention being compatible with a sprayable embodiment of thepresent invention are also unexpected.

“Low viscosity” in the context of this invention means that theviscosity measured at 20° C. with a rotational viscosimeter at a shearrate of 1800/s is not more than 50 mPa*s; in a preferred embodiment itis not more than 40 mPa*s, most preferred not more than 30 mPa*s.

“Water soluble” in the sense of this invention means that the solubilityof the polymers in water at 20° C. is not less than 0.5 weight percent,preferably not less than 1 weight percent and most preferably not lessthan 2 weight percent relative to the total composition. The solubilityis measured by stirring the respective amount of polymer in 100 ml waterfor 24 h and then visually judging the clarity of the solution.

“Stable” in the sense of this invention means both physical and chemicalstability: The pharmaceutical preparation preferably remains homogeneousduring storage at elevated temperatures (up to 50° C.) and whenrefrigerated (5° C.), it preferably shows no relevant loss of the activecomponent when stored at 25° C. for two years. In especially preferredembodiments, the preparation can be frozen at −20° C. and results in aclear solution after thawing and shaking of the container.

The pharmaceutical preparation of the present invention comprises 0.15to 3 weight percent of GTN.

The concentration of GTN is preferably in the range from 0.15 to 2weight percent, most preferred are concentrations of 0.2, 0.25, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 1.0, 1.2 and 1.5 weight percent.

Because of its explosive properties GTN for use in drug products ispreferably phlegmatised by the GTN producer. “Phlegmatised” means it isincorporated in a matrix reducing the dangerous characteristics. Bothliquid and solid compounds may be used as phlegmatisers. For example GTNcan be purchased as a 5% solution in propylene glycol, as a 10%trituration in lactose monohydrate or in 2.25% dilution in glucose. Whenthese concentrated GTN containing products are used directly forproducing preparations according to the invention the phlegmatisers arealso present in the pharmaceutical preparation. A preferred embodimentof the invention uses GTN in propylene glycol. Most preferably, a 5%solution of GTN in propylene glycol is used.

The pharmaceutical preparation according to this invention comprisesfrom 40 to 95 weight percent water. Preferably, the pharmaceuticalpreparation comprises from 60 to 90 weight percent of water. Preferredwater contents are 60, 65, 70, 75, 80, 85, 90, and 95 weight percents.

The pharmaceutical preparation comprises at least one water solublepolymer.

The water-soluble polymer is preferably a non-ionic water solublepolymer.

In a preferred embodiment, the water soluble polymer is selected fromthe group consisting of tyloxapol and poloxamer.

Tyloxapol is a non-ionic alkyl aryl polyether alcohol comprising thefollowing formula (I):

wherein m is preferably from 6 to 8 and n is preferably less than 6.Tyloxapol is available from e.g. Pressure Chemicals, Pittsburgh, Pa.,United States of America.

Poloxamer is a copolymer of ethylene oxide and propylene oxide andcomprises the following formula (II):

wherein a and b are preferably as defined below.

The poloxamer may be poloxamer 124, poloxamer 188, poloxamer 237,poloxamer 338 or poloxamer 407.

In poloxamer 124, a is from 10 to 15 and b is from 18 to 23. The contentof oxyethylene is 44.8% to 48.6% and the average relative molecular massis 2090 to 2360.

In poloxamer 188, a is from 75 to 85 and b is from 25 to 30. The contentof oxyethylene is 79.9% to 83.7% and the average relative molecular massis 7680 to 9510.

In poloxamer 237, a is from 60 to 68 and b is from 35 to 40. The contentof oxyethylene is 70.5% to 74.3% and the average relative molecular massis 6840 to 8830.

In poloxamer 338, a is from 137 to 146 and b is from 42 to 47. Thecontent of oxyethylene is 81.4% to 84.9% and the average relativemolecular mass is 12700 to 17400.

In poloxamer 407, a is from 95 to 105 and b is from 54 to 60. Thecontent of oxyethylene is 71.5% to 74.9% and the average relativemolecular mass is 9840 to 14600.

Poloxamer 407 is available e.g. under the trademark Kolliphor® P407 fromBASF, Ludwigshafen, Germany.

In a preferred embodiment the water soluble polymer is tyloxapol.

In a further preferred embodiment the water soluble polymer is poloxamer407.

In a further preferred embodiment the water soluble polymer is a mixtureof tyloxapol and at least one poloxamer.

The water soluble polymer is used in a concentration between 2 and 10weight percent, for example in a concentration of 2, 2.5, 3, 4, 5, 6, 7,8, 9 or 10 weight percent. The concentration is chosen as low aspossible, because with increasing amount of polymer the viscosity of thepreparation increases in a disproportionate manner.

The invention also encompasses a pharmaceutical preparation comprisingfrom 1.0 to 10 weight percent of at least one water soluble polymer and5 to 20 weight percent of ethanol. The water soluble polymer is, forexample in a concentration of 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10weight percent and ethanol is preferably in a concentration of 10, 12,15, 18 or 20 weight percent.

In preferred embodiments, the pharmaceutical preparation comprises:

-   -   from 1 to 5 weight percent of at least one water soluble polymer        and 10 to 20 weight percent of ethanol,    -   from 2 to 4 weight percent of at least one water soluble polymer        and 12 to 18 weight percent of ethanol, or    -   from 2 to 3 weight percent of at least one water soluble polymer        and 15 weight percent of ethanol.

The pharmaceutical preparations according to the invention mayadditionally comprise other pharmaceutically acceptable excipients. Thepharmaceutically acceptable excipient may be at least one additiveselected from the group consisting of preservatives, taste components,flavour components, sweeteners, acids and bases to adjust the pH andbuffering substances.

In an embodiment of the invention, the pharmaceutical preparationcomprises from 0 to 50 weight percent of at least one additive. In apreferred embodiment, the pharmaceutical preparation comprises from 30to 50 weight percent of at least one additive. In a more preferredembodiment, the pharmaceutical preparation comprises from 30 to 45weight percent of at least one additive.

In a further preferred embodiment of the invention, the pharmaceuticalpreparation comprises from 0 to 1 weight percent of at least oneadditive as defined above.

In one preferred embodiment the pharmaceutical preparation comprises thesugar alcohol xylitol as taste component.

It is desirable that the preparation causes a significant tasteperception in the patient to make him sure that e.g. the spray jet hasbeen actuated correctly and has hit the target area e.g. below thetongue. Xylitol is a “tooth friendly” sugar replacement. Hence, it isappropriate to use it in the pharmaceutical preparation for causing thedesired taste sensation.

In a preferred embodiment, the pharmaceutical preparation may be appliedas a spray jet that may be as small as 50 μl. Therefore, comparativelyhigh concentrations of xylitol would be necessary. Preferredconcentrations of xylitol in the pharmaceutical preparation aretherefore in the range of 5 to 50 weight percent, more preferred from 30to 45 weight percent.

The pharmaceutical preparation may additionally comprise at least onepreservative. Preservatives prevent the microbial deterioration of thepharmaceutical preparation. They may be used in concentrations from 0.01to 1 weight percent. Examples are benzoic acid, sorbic acid and themethyl, ethyl and propyl ester of 4-hydroxybenzoic acid. Beside thesepreservatives ethanol can also be used as a preservative. The necessaryamount of ethanol is in the range of 5 to 20 weight percent, preferablyfrom 10 to 20 weight percent.

In an embodiment of the invention, the pharmaceutical preparation doesnot comprise ethanol.

The stability of GTN in aqueous solution exhibits an optimum in theweakly acidic pH range. Therefore the buffering substance thatoptionally may be present in the preparation according to this inventionshall be chosen from the group of substances that possess a goodbuffering capacity, preferably in the pH range from 3.0 to 7.0.

In a preferred embodiment of the present invention, these bufferingsubstances are organic acids and their physiologically acceptable saltsor inorganic acids and their physiologically acceptable salts. Examplesof buffering substances are lactic acid/sodium lactate, citricacid/sodium citrate, gluconic acid/sodium gluconate, phosphoricacid/dibasic and monobasic potassium phosphate. The buffer concentrationis preferably chosen in a way that prevents pH shifts in the course of atwo-years storage at 25° C. of more than 0.5 pH units. Preferably it isbetween 0.1 and 1 weight percent.

In another preferred embodiment, the pharmaceutical preparation furthercomprises propylene glycol. The amount of propylene glycol can be in therange from 0 to 40 weight percent.

The viscosity of the pharmaceutical preparation is preferably ≤50 mPa*sand more preferably ≤30 mPa*s.

In a further aspect of the invention, the pharmaceutical preparation isused in a method for the prevention or treatment of an arterialinsufficiency.

According to the present invention, the term “treatment” or “prevention”means that not only symptoms of the disease are relieved but that alsothe disease itself is treated or prevented. In a preferred embodiment,the term “treatment” means improving the prognosis of said disease.

According to the invention, the term “arterial insufficiency” refers toany insufficient blood or oxygen supply or any other insufficient supplyof a tissue which is provided by an artery. This insufficient supply canbe overcome by the methods and uses of the present invention wherein apharmaceutical composition is used to increase the supply of a giventissue. The arterial insufficiency may occur both during physical restand during an exercise.

In a preferred embodiment of the present invention, the arterialinsufficiency is due to insufficient oxygen or blood supply of a tissuesupplied by the artery or a bypass or shunt during physical rest orexercise.

According to a further preferred embodiment, the arterial insufficiencyis due to an increased demand of oxygen or blood flow of a tissuesupplied by the artery or a bypass or shunt.

This increased demand of oxygen or blood flow can have several reasonsincluding but not limited to increased sport or physical activity, andincreased mental activity or a disease requiring an increased demand ofoxygen or blood flow.

According to a further preferred embodiment, the arterial insufficiencyis characterized by a partial (stenosis) or complete occlusion of anarterial vessel. In the context of the present invention, the term“partial occlusion” is equivalent to a stenosis.

The partial or complete occlusion of an arterial vessel is a well-knownphenomenon. It can have various reasons including, but not limited to,deposition of material in the blood vessels (includingnon-revascularisable stenoses), compression from external tissue orfluid next to the vessel (including disturbance in diastolic myocardialrelaxation), vascular spasm, dysfunction of the endothelium of thevessel resulting in a paradoxic vasoconstriction during exercise ormicrovascular impairment due to endothelial dysfunction or smooth musclecell abnormalities.

In a preferred embodiment, the arterial insufficiency is due to thedeposition of material in the blood vessels.

The deposition of materials in the blood vessels is a well-knownphenomenon resulting e.g. in atherosclerosis.

In a further preferred embodiment, the arterial insufficiency is due toan external or internal compression of an artery.

An internal compression of an artery may be due to an edema but also toa tumor putting pressure on the artery. Furthermore, this includes avasospastical constriction of the artery as e.g. in Prinzmetal's angina.In addition, this also includes the paradoxic vasoconstriction whiche.g. sometimes occur in an endothelial dysfunction or constricted smallarterial vessels due to endothelial or smooth muscle cell dysfunction.

An external compression may be due to an accident or any external forcewhich can put pressure on an artery.

In a further preferred embodiment, the arterial insufficiency is avascular disease.

According to a further preferred embodiment, the arterial insufficiencyis a disease selected from the group consisting of atherosclerosis, anischemic disease and a further chronic arterial disease.

In a further preferred embodiment, the arterial insufficiency is acoronary arterial insufficiency.

In a preferred embodiment, the coronary insufficiency is anatherosclerotic coronary arterial insufficiency, in particular coronaryartery disease (coronary heart disease or ischemic heart disease),stable angina pectoris, unstable angina pectoris, myocardial ischemia orchronic myocardial ischemia, acute coronary syndrome, myocardialinfarction (heart attack or ischemic myocardial infarction), orischemia-induced heart failure.

In a further preferred embodiment, the coronary insufficiency is anon-atherosclerotic, in particular coronary microvascular disease (smallvessel disease) or cardiac syndrome X, or Prinzmetal's angina.

In a further preferred embodiment, the arterial insufficiency is acerebral arterial insufficiency (intra- or extracranial).

In a preferred embodiment, the cerebral arterial insufficiency is anatherosclerotic cerebral arterial insufficiency, in particular cerebralischemia, extracranial carotid artery disease, extracranial vertebralartery disease, transient ischemic attack (mini stroke or pre-stroke),stroke, vascular dementia, ischemic brain diseases, or ischemiccerebrovascular disease.

The cerebral arterial insufficiency may also be ischemic microvascularbrain disease, small vessel vascular dementia, subcorticalarteriosclerotic encephalopathy (Binswanger's disease), Alzheimer'sdisease, or Parkinson's disease.

In a preferred embodiment, the arterial insufficiency is a peripheralarterial insufficiency.

In a preferred embodiment, the peripheral arterial insufficiency is anatherosclerotic peripheral arterial insufficiency, in particularperipheral vascular disease (peripheral artery disease (PAD) orperipheral artery occlusive disease (PAOD), including lower and upperextremity arterial disease), walking impairment or limited walkingdistance, claudication or intermittent claudication, ischemic limbsymptoms including pain, weakness, numbness or cramping in muscles dueto decreased blood flow, ischemic rest pain, abnormal ankle brachialpressure index, ischemic limb lesions (skin color changes, skin dryness,slowly or non-healing wounds or ulcers, necrosis, gangrene), chroniccritical limb ischemia, or the need for limb amputation.

In a preferred embodiment, the peripheral arterial insufficiency is anon-atherosclerotic peripheral arterial insufficiency, in particularRaynaud's syndrome (vasospasmatic), thrombangiitis obliterans(endangitis obliterans or Buerger's disease; recurring progressiveinflammation and thrombosis (clotting) of small and medium arteries andveins of the hands and feet), vascular inflammatory disease(vasculitis), or compartment syndromes.

In a preferred embodiment, the arterial insufficiency is induced bydiabetes, in particular diabetic ischemia disorders, including diabeticischemic foot syndrome, diabetic neuropathy, diabetic retinopathy andmaculopathy, or diabetic macular edema.

In a further preferred embodiment, the arterial insufficiency may be anintestinal arterial insufficiency, in particular an atheroscleroticintestinal arterial insufficiency, in particular ischemic bowel disease,mesenteric ischemia, or mesenteric infarction.

In a further preferred embodiment, the arterial insufficiency may be anurogenital arterial insufficiency, in particular an atheroscleroticurogenital arterial insufficiency, in particular erectile dysfunction,renal artery disease, renal ischemia, or renal infarction.

In a further preferred embodiment, the arterial insufficiency may be anerval arterial insufficiency, in particular tinnitus or diabeticneuropathy.

Furthermore, the arterial insufficiency may be in the context ofscleroderma (systemic sclerosis).

Furthermore, the arterial insufficiency may be in the context offibromuscular dysplasia.

In a preferred embodiment, the arterial insufficiency is a centralretinal artery insufficiency, in particular an atherosclerotic centralretinal artery insufficiency, in particular ocular arterialinsufficiency.

In a further preferred embodiment, the arterial insufficiency ischaracterized by an absence of an endothelial dysfunction.

The endothelial dysfunction is a well-known systemic pathological stateof the endothelium and can be broadly defined as an imbalance betweenvasodilating and vasoconstricting substances produced by or acting onthe endothelium.

In a further preferred embodiment, the arterial insufficiency is achronic arterial insufficiency. In the context of the present invention,the term “chronic arterial insufficiency” means that the course of thearterial insufficiency is chronic and often progredient.

According to a further embodiment, the chronic arterial insufficiencyincludes endothelial dysfunction, atherosclerosis, coronary arterydisease (coronary heart disease or ischemic heart disease), stableangina pectoris, coronary microvascular disease (small vessel disease)or cardiac syndrome X, Prinzmetal's angina, vascular dementia, ischemicbrain diseases, or ischemic cerebrovascular disease, ischemicmicrovascular brain disease, small vessel vascular dementia, subcorticalatherosclerotic encephalopathy (Binswanger's disease), Alzheimer'sdisease, Parkinson's disease, peripheral vascular disease (peripheralartery disease (PAD) or peripheral artery occlusive disease (PAOD),thrombangiitis obliterans (endangiitis obliterans or Buerger's disease),vascular inflammatory disease (vasculitis), fibromuscular dysplasia,diabetic ischemic disorders, diabetic neuropathy, ischemic boweldisease, erectile dysfunction, renal artery disease, tinnitus, andscleroderma (systemic sclerosis).

In a preferred embodiment, the arterial insufficiency is a diseaseand/or its symptom selected from the group consisting ofatherosclerosis, endothelial dysfunction, microvascular dysfunction,vasospastic disease, an ischemic disease, a further acute or chronicarterial disease, a microvessel disease, an intestinal arterialinsufficiency, an urogenital arterial insufficiency, a nerval arterialinsufficiency, scleroderma, or a central retinal artery insufficiency.

In a further preferred embodiment, the arterial insufficiency is acoronary arterial insufficiency preferably selected from the groupconsisting of coronary artery disease (coronary heart disease), ischemicheart disease, stable and unstable angina pectoris, acute coronarysyndrome, myocardial ischemia, myocardial infarction, ischemia-inducedheart failure, coronary microvascular disease (small vessel disease) orcardiac syndrome X, coronary spasms or Prinzmetal's angina.

In a further preferred embodiment, the arterial insufficiency is anintra- or extracranial cerebral arterial insufficiency preferablyselected from the group consisting of cerebral ischemia, extracranialcarotid artery disease, extracranial vertebral artery disease, transientischemic attack (mini-stroke or pre-stroke), stroke, vascular dementia,ischemic brain diseases, or ischemic cerebrovascular disease.

In a most preferred embodiment, the arterial insufficiency is aperipheral arterial insufficiency, preferably selected from the groupconsisting of Raynaud's syndrome, diabetic ischemia disorders, includingdiabetic ischemic foot syndrome or diabetic neuropathy, thromboangiitisobliterans (endangiitis obliterans or Buerger's disease), vascularinflammatory disease (vasculitis), compartment syndromes, peripheralartery disease (peripheral artery occlusive disease), walking impairmentor limited walking distance, claudication or intermittent claudication,ischemic limb symptoms (pain, weakness, numbness or cramping in musclesdue to decreased blood flow), ischemic rest pain, abnormal anklebrachial pressure index, ischemic limb lesions (skin color change, skindryness, slowly or non-healing wounds or ulcers, necrosis, gangrene),chronic critical limb ischemia, or the need for limb amputation.

The pharmaceutical preparation according to the present invention isparticularly suitable for promoting collateral circulation. This mightbe explained by the short-term dilatation of the collateral vesselinduced by the pharmaceutical preparation which, in turn, has asignificantly improved effect on arteriogenesis. An arteriogenesis e.g.can function as a good prophylaxis of tissue ischemia in general or ofsevere disease associated events as e.g. myocardial infarction, stroke,ischemic heart failure, such as post-infarction heart failure, or limbamputation in the case of peripheral artery disease. Arteriogenesis canhave a positive influence on the prognosis of the disease and preventprogredient disease development.

According to the invention, the pharmaceutical preparation isadministered in an amount capable of inducing arteriogenesis. Theskilled person will appreciate that this amount will depend on thesubject to which the pharmaceutical preparation is administered.Generally, the amount of active substance to be administered may be from0.05 to 50 mg per day, but this can vary due to the weight of thesubject, its hemodynamic response to the pharmaceutical preparationand/or the severity of the disease.

The amount to be administered can also vary depending on the way ofadministration. E.g. when administered sublingually, inhalatively, orbucally the preferred amount of GTN can be from 0.1 to 8 mg per day.E.g. when administered dermally, the preferred amount can be 0.1 to 30mg

Hence, the amount of the pharmaceutical preparation to be administeredmay be from 0.1 to 15 ml per day, or, alternatively, from 0.02 to 3 mlper day, dependent on the nature of the pharmaceutical preparationand/or its way of administration.

In a preferred embodiment, an amount of 0.05 to 1.6 mg GTN may beadministered per single application lingually, sublingually, bucally ororomucosally. This amount may be administered 1- up to 5-timesmaximally, resulting in a maximal dosage of active substance of 8 mg perday.

In a preferred embodiment, an amount of 0.025 to 1.0 ml (0.025, 0.05,0.1, 0.2, 0.3, 0.5, 0.75, 1.0) of the pharmaceutical preparation isapplied for at least 1- up to maximal 5-times daily, resulting in amaximal dosage of active substance of 3.2 mg per day.

According to the invention, the term “administration of thepharmaceutical preparation” means that a given dosage of thepharmaceutical preparation is administered. Depending on the way ofadministration, the skilled person will appreciate that theadministration may take some time. In a preferred embodiment, thepharmaceutical preparation is administered in form of a spray, sprayableor injectable solution, or inhalable aerosol, which means that theadministration may be completed within seconds. However, theadministration of the pharmaceutical preparation may also take longer,e.g. if the pharmaceutical preparation is administered to the patient byway of infusion. Modes of administration of the pharmaceuticalpreparation are further discussed below.

Furthermore, according to the invention, the pharmaceutical preparationis administered in a manner capable of inducing arteriogenesis.

The inventors of the present invention have surprisingly found out thata pharmaceutical preparation according to the invention is capable ofinducing arteriogenesis when administered in an intermitting manner.

According to the invention, the term “intermitting manner” means thatthe pharmaceutical preparation is administered in a way that its plasmaor tissue levels are only elevated in a short-term manner after theadministration of the pharmaceutical preparation but then again decline.This can be achieved for example if the pharmaceutical preparation has acomposition as defined above and the administration of thepharmaceutical preparation is followed by a time period withoutadministration and then the pharmaceutical preparation is againadministered to the subject. Furthermore, this way of administrationavoids that the subject is developing tolerances against thepharmaceutical preparation and that the subject is developingendothelial dysfunction.

The induction of endothelial dysfunction is a parameter which has aprognostic significance in patients with coronary artery disease. Thedevelopment of tolerances as well as the induction of endothelialdysfunction are well known disadvantages caused by the sustained, longterm exposure to NO donors (Uxa A. et al., Journal of CardiovascularPharmacology, 2010, 56 (4): 354-359).

Moreover, the administration of a pharmaceutical preparation accordingto the invention in an intermitting manner has the effect that it mimicsthe physiological situation of the organism as, for example, comparableto the endogenous release of NO upon physical training. In other words,the pharmaceutical preparation of the present invention acts as abiomimetic when applied in an intermitting manner.

In a preferred embodiment, the plasma or tissue levels of the activecompound of the pharmaceutical preparation are elevated for not morethat 240, 180, 120, or 60 minutes, or for not more than 50, 40, 30, 15,10 or 5 minutes.

Furthermore, this also implies that the pharmaceutical preparation canbe administered in chronic manner, i.e. without taking account ofdisease developments implying an acute treatment with the pharmaceuticalpreparation. Furthermore, it also implies that a therapy plan can beestablished without taking account of disease developments implying anacute treatment with the pharmaceutical preparation.

The pharmaceutical preparation is used to achieve a relief or acute(i.e. immediate) prevention of the symptoms of a corresponding disease.These symptoms for example include pain and/or dyspnea in the case of acardiovascular disease, and the relief or acute prevention of thesymptoms was achieved by vasodilation and resulting pain and/or dyspnearelief.

In the context of the present invention, the term “intermittently” alsomeans that the pharmaceutical preparation is not administeredcontinuously, for example by means of long term intravenous infusion orwith the help of an implanted pump which constantly delivers thepharmaceutical preparation to the subject. Rather, this term also meansthat there is an interval between two administrations of thepharmaceutical preparation, and that the pharmaceutical preparation isgiven several times, e.g. at least 1, 2, 3, 4, 5, 6, 8, 9, 12 or 16times a day.

As the skilled person will appreciate, one administration of thepharmaceutical preparation may include an administration in one or moredosage forms, e.g. hubs (puffs) in case of a spray. For example, oneadministration may include the administration of one to three hubs(puffs).

As to the schedule of administration, the skilled person will appreciatethat there are many ways to achieve this intermitting administration.For example, it is possible to administer the pharmaceutical preparationat least once a day and at least on one day a week for at least twoweeks. However, it is equally possible to administer the pharmaceuticalpreparation for only one week if the pharmaceutical preparation isadministered several times during this week.

Preferable, the pharmaceutical preparation is administered once, twiceor three times a day, wherein even more preferred the time periodbetween two administrations of the pharmaceutical preparation is atleast 4 hours, in particular 8 hours, in particular at least 10 hours or12 hours.

Although possible, it is not necessary that the time periods between twoadministrations of the pharmaceutical preparations are the same. Rather,it is preferred that these time periods differ, depending on theindividual administration schedule.

In an embodiment, the pharmaceutical preparation is administered atleast on one day a week. However, the pharmaceutical preparation mayalso be administered on 2, 3, 4, 5, 6 or 7 days a week. In an especiallypreferred embodiment, the pharmaceutical preparation is administered atleast on 3 or 4 days a week.

According to the invention, it is possible to administer thepharmaceutical preparation for a period of several weeks or months. Thisis particularly preferred in order to induce arteriogenesis efficiently,although also a shorter administration of one of two weeks is possible.

In an embodiment, the pharmaceutical preparation is administered for 2to 8 weeks. It is equally preferred to administer the pharmaceuticalpreparation for 3 to 6, 3 to 8, 3 to 10 or 4 to 8, 4 to 10 or 4 to 12weeks. These numbers are only examples and may vary depending on theindividual schedule of the subject.

In an embodiment, the pharmaceutical preparation is taken at least oncea week for at least 8 weeks, in particular for at least 12 weeks.

In a further preferred embodiment, the pharmaceutical preparation istaken not longer than 6, 8 or 12 months. However, it is also possible totake the pharmaceutical preparation for 2, 3 or even more years.Furthermore, it is also possible that the pharmaceutical preparation isadministered for decades or even through the whole life of the subject.

In the context of such long-term administrations, it is preferred thatthe pharmaceutical preparation is administered once or twice a week orat least once or twice a week.

It has been described previously that an exogenous stimulation ofpulsatile shear forces in an individual may result in arteriogenesis.Furthermore, it has been described how the pulsatile shear forces can bemeasured (WO 2010/072416).

Consequently, in a preferred embodiment, the pharmaceutical preparationis administered in conjunction with an exogenous stimulation of thepulsatile shear forces in the artery.

In a further preferred embodiment, the pharmaceutical preparation isadministered when the subject is at rest.

With respect to said embodiment of the invention, the pharmaceuticalpreparation should be administered in a way that it is active in thebody of the subject when the exogenous stimulation is applied. In thiscontext, active means that either the NO release is not yet terminatedor the NO released from the active substance GTN is still present andactive. Depending on the physiological halftime of the active substancein the subject and its formulation, the skilled person will be capableof determining when the pharmaceutical preparation has to beadministered to the subject in order to ensure that it is active uponthe exogenous stimulation.

In the case of GTN, the halftime and its persistence in the body of thesubject has been intensively studied, e.g. after intravenous orsublingual application, where it is 2 to 5 minutes in the blood plasma,see e.g. Armstrong P. W. et al., Circulation, 1979, 59: 585-588 orArmstrong P. W. et al., Circulation, 1980, 62:160-166.

In general, the halftime of GTN in the blood plasma is 2 to 5 minutes.

It is to be understood that, in the context of the present invention,the term “halftime” refers to the half-life and/or to the half-life timeof the GTN in the subject's body, in particular in the subject's bloodplasma.

In an embodiment, the pharmaceutical preparation is administered in thetime period of 30 minutes before the onset of the exogenous stimulationuntil 30 minutes after the termination of the exogenous stimulation.

More preferably, the pharmaceutical preparation is administered in thetime period of 15 minutes, preferably 5 minutes, more preferably 2minutes before the exogenous stimulation until 30, preferably 15, morepreferably 5 minutes after the onset of the exogenous stimulation.

In a further preferred embodiment, the pharmaceutical preparation isadministered once a day, five times a week for 6 weeks 2-5 minutesbefore the exogenous stimulation.

The exogenous stimulation of the pulsatile shear forces may be achievedby any known way. This includes an stimulation with the help ofmedicaments like medicaments which increase the blood pressure.

In a preferred embodiment, said stimulation is achieved by physicalexercise or the application of an endogenous force to the arterialvessel.

According to the invention, the term “physical exercise” means anytraining of the subject, including but not limited to training inexercise rooms, jogging, walking, nordic walking, swimming, dancing,cycling and hiking. The skilled person will appreciate that any exercisewill be helpful in the context of the invention, provided that it isperformed in conjunction with the administration of the pharmaceuticalpreparation. Preferably, the term “physical exercise” does not includeunsupervised, unprescribed routine movements like casual walking orhouse work.

As discussed above, it has been found in the context of the presentinvention that the pharmaceutical preparation is capable of inducingarteriogenesis. This enables not only the treatment of an alreadyexisting disease. Rather, in the context of the present invention, it isalso possible to prevent the disease. Consequently, in a preferredembodiment of the present invention, the method aims at the preventionof said arterial insufficiency.

In the context of the present invention, it has been possible to reducethe infarct size in case of an already existing occlusion. Furthermore,it has been possible to reduce arrhythmias in the subjects.Consequently, in a preferred embodiment of the present invention, themethod results in a reduction of the infarct size, in reducedarrhythmias or in a decreased ST segment elevation.

The pharmaceutical preparation can be administered in any suitable wayso that it can be incorporated into the subject. This includes an oral,parenteral or intravenous administration as well as the injection of thepharmaceutical preparation into the body of the subject, but also anadministration to a mucous membrane or the skin of the subject.

In a most preferred embodiment of the present invention, thepharmaceutical preparation is administered orally or sublingually.

In a preferred embodiment of the present invention, the pharmaceuticalpreparation is administered topically, lingually, sublingually,inhalatively, bucally, mucosally, transmucosally or oromucosally,dermally or cutaneously, transdermally or percutaneously.

The term “mucosally” according to the present invention means that thepharmaceutical preparation is applied on the mucosa.

The term “transmucosally” according to the present invention means thatthe pharmaceutical preparation, especially the active substance, passesthe mucosa.

The term “oromucosally” according to the present invention means thatthe pharmaceutical preparation, especially the active substance, isapplied in the oral cavity and/or the throat.

The terms “dermally” and “cutaneously” as well as “transdermally” and“percutaneously” can be used interchangeably in the present invention.

In case of a lingual, sublingual or oromucosal administration, it ispreferred that the pharmaceutical preparation is administered with thehelp of a spray, sprayable or injectable solution, or by an inhalatordevice, from which the pharmaceutical preparation can be easily inhaledand adsorbed. It is equally preferred that the pharmaceuticalpreparation is administered in the form of an inhalable gas, or aerosol.

The pharmaceutical preparation can be formulated in any suitable way forthe above mentioned administration modes. Such formulations are known tothe person skilled in the art and include the formulation in suitablebuffers or as an aerosol.

In a preferred embodiment, the pharmaceutical preparation is formulatedin a way that allows a fast release of the active substance from theformulation. This includes e.g. formulations which do not hold back theactive substance for a longer time period, but which release the activesubstance within e.g. 45, 30 or 15, 10, 5 minutes or 1 minute.

Through the invention, it is preferred that the subject to which thepharmaceutical preparation is applied is a human subject.

In a further aspect, the present invention also relates to apharmaceutical preparation for use in a method for the prevention ortreatment of an arterial insufficiency, wherein the pharmaceuticalpreparation is administered in an amount and manner effective for theinduction of arteriogenesis.

All features and preferred embodiments discussed above for the method oftreating or preventing an arterial insufficiency also apply to thepharmaceutical preparation for use according to this aspect of theinvention.

In another aspect, the present invention also relates to a method of thesuppression of negative effects associated with any treatment of anarterial insufficiency which is anti-arteriogenic or inhibitingarteriogenesis, comprising administering to a subject subjected to saidtreatment a pharmaceutical preparation in an amount and manner effectivefor the induction of arteriogenesis.

In a preferred embodiment, said treatment is an acetyl salicylic acid(ASA), glycoproteinIIbIIIa antagonists, or etanercept (soluble tumornecrosis factor alpha receptor) treatment.

It is known in the art that ASA is an inhibitor of arteriogenesis(Singer E. et al., Vasa, 2006, 35 (3): 174-177). Consequently, the ASAtreatment of cardiovascular diseases, although being a standard therapy,has significant side effects and disadvantages. In the context of thepresent invention, it has been found that the pharmaceuticalpreparations are capable of overcoming the negative effects associatedwith an ASA treatment (see example section). Based on these findings,the inventors conclude that also the negative side effects associatedwith other medications like glycoproteinIIbIIIa antagonists oretanercept treatment can also be diminished.

Furthermore, the present invention also relates to a pharmaceuticalpreparation for use in a method of the suppression of negative effectsassociated with any treatment of an arterial insufficiency which isanti-arteriogenic or inhibiting arteriogenesis, wherein thepharmaceutical preparation is administered to a subject subjected tosaid treatment in an amount and manner effective for the induction ofarteriogenesis.

In a preferred embodiment, said treatment is an acetyl salicylic acid(ASA), glycoproteinIIbIIIa antagonists, or etanercept (soluble tumornecrosis factor alpha receptor) treatment.

All features and preferred embodiments discussed above for the method oftreating or preventing an arterial insufficiency also apply to themethod for the suppression of negative effects according to this aspectof the invention or to said pharmaceutical preparation for use accordingto this aspect of the invention.

In a further aspect, the present invention also relates to a method forthe prevention or treatment of a cardiac arrhythmia, wherein apharmaceutical preparation according to the invention is administered toa subject in an amount and manner effective for the treatment of saidcardiac arrhythmia. Furthermore, the present invention also relates to apharmaceutical preparation for use in a method for the prevention ortreatment of a cardiac arrhythmia, wherein the pharmaceuticalpreparation is administered to a subject in an amount and mannereffective for the treatment of said cardiac arrhythmia.

In the context of the present invention, the inventors have found thatpharmaceutical preparations according to the invention are capable toprevent and treat arrhythmias.

All features and embodiments defined above with respect to thepharmaceutical preparation and its formulation and administration alsoapply to this method or pharmaceutical preparation for use according tothe invention.

The present invention also relates to a method of promoting collateralcirculation comprising the step of exposing a subject to atherapeutically effective amount of a pharmaceutical preparationaccording to the invention wherein the therapeutically effective amountof the pharmaceutical preparation promotes arteriogenesis sufficient toaugment collateral circulation in a physiological or pathologicalcondition.

The term collateral circulation describes the circulation of bloodthrough so-called collateral vessels. These vessels are smallarterioles, which are part of a network that interconnects perfusionterritories of arterial branches. In the case that the main arteryitself is not capable of sufficiently supplying a tissue, e.g. due to anarterial occlusion, these collateral vessels are recruited and candevelop to large conductance arteries, to bypass the site of an arterialocclusion and/or to compensate blood flow to ischemic territoriessupplied by the or insufficient artery. In the context of the presentinvention, the promotion of collateral circulation occurs viaarteriogenesis.

According to the invention, the term “physiological condition” denotesany condition of the subject which is not related to any disease.

According to the invention, the term “pathological condition” denotesany condition of the subject which is related to a disease.

Preferably, the subject suffers from an arterial insufficiency.

All features and preferred embodiments discussed above for the method oftreating or preventing an arterial insufficiency also apply to themethod of promoting collateral circulation.

With respect to the aspects defined above where the pharmaceuticalpreparation is administered in a manner sufficient to inducearteriogenesis this manner is preferably an intermitting manner asdefined above.

Another aspect of the present invention is a process for the preparationof a pharmaceutical preparation, comprising admixing components (a) and(c1) in water or admixing components (a) and (c2) in water.

The production process of the pharmaceutical preparations according tothis invention is straight forward. Generally all water solublecompounds are dissolved in water, whereby it may be appropriate todissolve the water soluble polymer in a separate first step. Then theGTN, preferably in the form of a concentrate, is added and the mixtureis stirred vigorously until a homogeneous solution is formed.

Another aspect of the present invention is a kit comprising thepharmaceutical preparation according to the invention, wherein the kitis a spray. This kit may consist of container and spray pump. Thecontainer may be a glass or plastic bottle.

Preferred embodiments of the pharmaceutical preparations and ofpreparing the same are:

In a first embodiment 2 to 5 weight percent of poloxamer 407 aredissolved in water. Then 4 to 12 weight percent GTN in propylene glycol(5%) are added and stirred for 15 minutes. Optionally a mixture of 0.02%of propyl 4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoate isadded.

In a second embodiment 30 to 50 weight percent of xylitol and 2 to 5weight percent of poloxamer 407 are dissolved in water. Then 4 to 12weight percent GTN in propylene glycol (5%) are added and stirred for 15minutes. Optionally a mixture of 0.02% of propyl 4-hydroxybenzoate and0.08% of methyl 4-hydroxybenzoate is added.

In another embodiment 30 to 50 weight percent of xylitol and 1.5 to 5weight percent of tyloxapol are dissolved in water. Then 4 to 12 weightpercent GTN in propylene glycol (5%) are added and stirred for 15minutes. Optionally a mixture of 0.02% of propyl 4-hydroxybenzoate and0.08% of methyl 4-hydroxybenzoate is added.

In yet another embodiment 30 to 50 weight percent of xylitol, 1 to 5weight percent of tyloxapol and 1 to 5 weight percent of poloxamer 407are dissolved in water. Then 4 to 12 weight percent GTN in propyleneglycol (5%) are added and stirred for 15 minutes. Optionally a mixtureof 0.02% of propyl 4-hydroxybenzoate and 0.08% of methyl4-hydroxybenzoate is added.

In one embodiment 30 to 50 weight percent of xylitol and 1 to 5 weightpercent of poloxamer 407 are dissolved in water. Then 4 to 12 weightpercent GTN in propylene glycol (5%) are diluted in 10 to 20 weightpercent ethanol. Both solutions are combined and stirred for 15 minutes.Alternatively, the xylitol can be omitted.

A similar preparation free of propylene glycol can be achieved bydissolving 30 to 50 weight percent of xylitol and 1 to 5 weight percentof poloxamer 407 in water, adding 4 to 12 weight percent GTN in ethanol(5%) and 8 to 16 weight percent ethanol, and stirring for 15 minutes.

In a further embodiment 2 to 10 weight percent of poloxamer 407 aredissolved in water. Then 4 to 12 weight percent GTN in propylene glycol(5%) are added and stirred for 15 minutes. Optionally a mixture of 0.02%of propyl 4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoate or0.1% benzoic acid as preservative is added.

In another embodiment 30 to 50 weight percent of xylitol and 2 to 10weight percent of poloxamer 407 are dissolved in water. Then 4 to 12weight percent GTN in propylene glycol (5%) are added and stirred for 15minutes. Optionally a mixture of 0.02% of propyl 4-hydroxybenzoate and0.08% of methyl 4-hydroxybenzoate or 0.1% benzoic acid as preservativeis added.

In an embodiment 1.5 to 7 weight percent of tyloxapol are dissolved inwater. Then 4 to 12 weight percent GTN in propylene glycol (5%) andoptionally a mixture of 0.02% of propyl 4-hydroxybenzoate and 0.08% ofmethyl 4-hydroxybenzoate or 0.1% benzoic acid as preservative(s) areadded and the mixture is stirred for 15 minutes.

In another embodiment 30 to 50 weight percent of xylitol and 1.5 to 7weight percent of tyloxapol are dissolved in water. Then 4 to 12 weightpercent GTN in propylene glycol (5%) are added and stirred for 15minutes. Optionally a mixture of 0.02% of propyl 4-hydroxybenzoate and0.08% of methyl 4-hydroxybenzoate or 0.1% benzoic acid aspreservative(s) is added.

In a further embodiment 1 to 5 weight percent of tyloxapol and 1 to 10weight percent of poloxamer 407 are dissolved in water. Then 4 to 12weight percent GTN in propylene glycol (5%) and optionally a mixture of0.02% of propyl 4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoateor 0.1% benzoic acid as preservative(s) are added and the mixture isstirred for 15 minutes.

In yet another embodiment 30 to 50 weight percent of xylitol, 1 to 5weight percent of tyloxapol and 1 to 10 weight percent of poloxamer 407are dissolved in water. Then 4 to 12 weight percent GTN in propyleneglycol (5%) are added and stirred for 15 minutes. Optionally a mixtureof 0.02% of propyl 4-hydroxybenzoate and 0.08% of methyl4-hydroxybenzoate or 0.1% benzoic acid as preservative(s) is added.

In general, formulas containing xylitol are more appropriate for oral orsublingual application, whereas those not containing any sugar alcoholare better for dermal application.

A first embodiment comprises 2 to 5 weight percent of poloxamer 407dissolved in water, 4 to 12 weight percent GTN in propylene glycol (5%)and optionally a mixture of 0.02% of propyl 4-hydroxybenzoate and 0.08%of methyl 4-hydroxybenzoate or 0.1% benzoic acid as preservative(s).

A second embodiment comprises 2 to 5 weight percent of poloxamer 407dissolved in water, 30 to 50 weight percent of xylitol, 4 to 12 weightpercent GTN in propylene glycol (5%) and optionally a mixture of 0.02%of propyl 4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoate or0.1% benzoic acid as preservative(s).

Another embodiment comprises 1.5 to 5 weight percent of tyloxapoldissolved in water, 30 to 50 weight percent of xylitol, 4 to 12 weightpercent GTN in propylene glycol (5%) and optionally a mixture of 0.02%of propyl 4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoate or0.1% benzoic acid as preservative(s).

Yet another embodiment comprises 1 to 5 weight percent of tyloxapol and1 to 5 weight percent of poloxamer 407 dissolved in water, 30 to 50weight percent of xylitol, 4 to 12 weight percent GTN in propyleneglycol (5%) and optionally a mixture of 0.02% of propyl4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoate or 0.1% benzoicacid as preservative(s).

Further embodiments comprise 1 to 5 weight percent of poloxamer 407dissolved in water, 30 to 50 weight percent of xylitol, 4 to 12 weightpercent GTN in propylene glycol (5%) and 10 to 20 weight percentethanol. Alternatively, the xylitol can be omitted.

A further embodiment comprises 2 to 10 weight percent of poloxamer 407dissolved in water, 4 to 12 weight percent GTN in propylene glycol (5%)and optionally a mixture of 0.02% of propyl 4-hydroxybenzoate and 0.08%of methyl 4-hydroxybenzoate or 0.1% benzoic acid as preservative(s).

Another embodiment comprises 2 to 10 weight percent of poloxamer 407dissolved in water, 30 to 50 weight percent of xylitol, 4 to 12 weightpercent GTN in propylene glycol (5%) and optionally a mixture of 0.02%of propyl 4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoate or0.1% benzoic acid as preservative(s).

An embodiment comprises 1.5 to 7 weight percent of tyloxapol dissolvedin water, 4 to 12 weight percent GTN in propylene glycol (5%) andoptionally a mixture of 0.02% of propyl 4-hydroxybenzoate and 0.08% ofmethyl 4-hydroxybenzoate or 0.1% benzoic acid as preservative(s).

Another embodiment comprises 1.5 to 7 weight percent of tyloxapoldissolved in water, 30 to 50 weight percent of xylitol, 4 to 12 weightpercent GTN in propylene glycol (5%) and optionally a mixture of 0.02%of propyl 4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoate or0.1% benzoic acid as preservative(s).

A further embodiment comprises 1 to 5 weight percent of tyloxapol and 1to 10 weight percent of poloxamer 407 dissolved in water, 4 to 12 weightpercent GTN in propylene glycol (5%) and optionally a mixture of 0.02%of propyl 4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoate or0.1% benzoic acid as preservative(s).

Yet another embodiment comprises 1 to 5 weight percent of tyloxapol and1 to 10 weight percent of poloxamer 407 dissolved in water, 30 to 50weight percent of xylitol, 4 to 12 weight percent GTN in propyleneglycol (5%) and optionally a mixture of 0.02% of propyl4-hydroxybenzoate and 0.08% of methyl 4-hydroxybenzoate or 0.1% benzoicacid as preservative(s).

Further embodiments comprise 1 to 5 weight percent of poloxamer 407dissolved in water, 30 to 50 weight percent of xylitol, 4 to 12 weightpercent GTN in propylene glycol (5%) and 10 to 20 weight percentethanol. Alternatively, the xylitol can be omitted.

Embodiments free of propylene glycol comprise 30 to 50 weight percent ofxylitol and 1 to 5 weight percent of poloxamer 407 dissolved in water, 4to 12 weight percent GTN in ethanol (5%) and 8 to 16 weight percentethanol. Also in these embodiments xylitol can be omitted preferablywhen the preparation is designed for a dermal application.

The invention is further described by the attached figures and examples,which are intended to illustrate, but not to limit the invention.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Beaker comprising a composition according to comparative example1.

FIG. 2: Beaker comprising a composition according to comparative example3.

FIG. 3: Course of the ST segment elevation per beat after FPO (=finalocclusion to induce infarct) of 5- and 10-days-control-groups. ECG graphin middle grey indicates 5 DAYS RIP PBS, n=8: 0.104±0.016 mV; ECG graphin black indicates 5 DAYS SHAM PBS, n=8: 0.134±0.034 mV; ECG graph inlight grey indicates 10 DAYS RIP PBS, n=7: 0.055±0.033 mV; ECG graph indark grey indicates 10 DAYS SHAM PBS, n=7: 0.124±0.039 mV.

ECG was recorded 90 minutes after FPO. Course of the ST segmentelevation per beat at first 1200 beats revealed no differences between5- and 10-days-sham-groups and 5-days-RIP-group. Only in the10-days-RIP-group a lower ST segment elevation was observed.

FIG. 4: ST segment elevation of 5- and 10-days-control-groups. Column 1shows ST segment elevation of 5 DAYS SHAM PBS group; column 2 shows STsegment elevation of 5 DAYS RIP PBS group; column 3 shows ST segmentelevation of 10 DAYS SHAM PBS group; column 4 shows ST segment elevationof 10 DAYS RIP PBS group; standard deviation is indicated in error bars;asterisk indicates significant compared to 10 DAYS SHAM PBS (nominal pvalue<0.025); double asterisk indicates significant compared to 5 DAYSRIP PBS (nominal p value<0.025).

Diagram shows mean of ST segment elevation maximum per group. After 5days there was no significant difference found between RIP and SHAM.After 10 days in the RIP group ST segment elevation maximum wassignificantly lower compared to sham (*) and 5-day RIP control (**) (*,**nominal p-value<0.025).

FIG. 5: Course of the ST segment elevation per beat after FPO (module 1:Sham operation without the RIP). ECG graph in black indicates 5 DAYSSHAM PBS, n=8: 0.134±0.034 mV; ECG graph in light grey indicates 5 DAYSSHAM NTG, n=7: 0.124±0.058 mV; ECG graph in middle grey indicates 5 DAYSSHAM NTG-PLACEBO, n=6: 0.131±0.043 mV.

The course of the ST segment elevation per beat after FPO revealed nodifferences between sham control and treated groups after 5 days.

FIG. 6: ST segment elevation (module 1: Sham operation without the RIP).Column 1 shows 5 DAYS SHAM PBS; column 2 shows 5 DAYS SHAM NTG-Placebo;column 3 shows 5 DAYS SHAM NTG; standard deviation is indicated by errorbars.

Diagram shows mean of ST segment elevation maximum per group. Nodifference in ST segment elevation maximum was found between shamcontrol and treated groups.

FIG. 7: Course of the ST segment elevation per beat after FPO (module 2:NO intermittent (NTG)). ECG graph in light grey indicates 5 DAYS RIPPBS, n=8: 0.104±0.016 mV; ECG graph in middle grey indicates 5 DAYSNTG-PLACEBO, n=6; 0.096±0.061 mV; ECG graph in black indicates 5 DAYSRIP NTG, n=7: 0.052±0.030 mV.

Compared to control treatment with PBS or NTG-Placebo a lower ST segmentelevation course was detected after NTG treatment 5 days after RIP.

In the NTG group (“5 DAYS RIP NTG”) ST segment elevation issignificantly decreased compared to the PBS and NTG-Placebo group. Thereis no significance between the PBS and NTG-PLACEBO-group.

FIG. 8: ST segment elevation (module 2: NO intermittent (NTG)). Column 1shows 5 DAYS RIP PBS; column 2 shows 5 DAYS NTG-PLACEBO; column 3 shows5 DAYS RIP NTG; standard deviation is indicated by error bars, asteriskindicates significant decrease of ST segment elevation compared to PBSand NTG-Placebo group (nominal p-value<0.017).

Diagram shows mean of ST segment elevation maximum per group. Aftertreatment with NTG, the ST segment elevation maximum was significantlydecreased compared to PBS and NTG-Placebo treatment 5 days after RIP(nominal p-value<0.017).

FIG. 9: Course of the ST segment elevation per beat after FPO (module 3:NO continuous (ISDN retard)). ECG graph in light grey indicates 5 DAYSRIP PBS, n=8: 0.104±0.016 mV; ECG graph in middle grey indicates 5 DAYSISDN-PLACEBO, n=7: 0.110±0.069 mV; ECG graph in black indicates 5 DAYSRIP ISDN, n=7: 0.062±0.027 mV.

Compared to control treatment with PBS or ISDN-Placebo a lower STsegment elevation course was detected after ISDN treatment 5 days afterRIP.

ST segment elevation in the ISDN group (“5 DAYS RIP ISDN”) is decreasedcompared to the PBS group but there is no significance as well asbetween the PBS and ISDN-PLACEBO-group.

FIG. 10: ST segment elevation (module 3: NO continuous (ISDN retard)).Column 1 shows 5 DAYS RIP PBS; column 2 shows 5 DAYS RIP ISDN-PLACEBO;column 3 shows 5 DAYS RIP ISDN; standard deviation is indicated by errorbars.

Diagram shows mean of ST segment elevation maximum per group. Aftertreatment with ISDN, the ST segment elevation maximum wasnon-significantly decreased compared to PBS and ISDN-Placebo treatment 5days after RIP.

FIG. 11: Course of the ST segment elevation per beat after FPO (module4: NO intermittent plus ASA). ECG graph in light grey indicates 5 DAYSRIP PBS, n=8; 0.104±0.016 mV; ECG graph in middle grey indicates 5 DAYSRIP ASA+PBS, n=7: 0.138±0.098 mV; ECG graph in dark grey indicates 5DAYS RIP ASA+NTG-PLACEBO, n=6: 0.144±0.091 mV; ECG graph in blackindicates 5 DAYS RIP NTG+ASA, n=7: 0.088±0.071 mV.

Treatment with NTG+ASA was compared to with PBS+ASA, NTG-Placebo+ASA andPBS. In general, all curves overlay at the same range.

ST segment elevation in the group treated with PBS and ASA is highercompared to the PBS control group, but there is no significance as wellas between the ASA+NTG-PLACEBO-group. In the ASA+NTG-group ST segmentelevation is decreased compared to the group treated with ASA and PBS.

FIG. 12: ST segment elevation (module 4: NO intermittent plus ASA).Column 1 shows 5 DAYS RIP PBS; column 2 shows 5 DAYS RIP PBS+ASA; column3 shows 5 DAYS RIP NTG-PLACEBO; column 4 shows 5 DAYS RIPNTG-PLACEBO+ASA; column 5 shows 5 DAYS RIP NTG; column 6 shows 5 DAYSRIP NTG+ASA; standard deviation is indicated by error bars.

Diagram shows mean of ST segment elevation maximum per group. Treatmentwith NTG+ASA was compared to PBS+ASA, NTG-Placebo+ASA and PBS.Furthermore, all ASA groups (PBS+ASA, NTG-Placebo+ASA, NTG+ASA) werecompared to their controls (PBS, NTG-Placebo, NTG). No significantdifferences were detected.

FIG. 13: Arrhythmias during FPO (module 1: Sham Operation (without theRIP)). Numbers of columns are given in consecutive order of the columnsin group IVb. Column 1 shows 5 DAYS SHAM PBS; column 2 shows 5 DAYS SHAMNTG-PLACEBO; column 3 shows 5 DAYS SHAM NTG.

In accordance with Lown classification, all sham groups werepredominantly scaled into grade IVb.

In the “5 DAYS SHAM PBS” group 87.5% of the rats have class IVbarrhythmias and 12.5% class IVa. In the “5 DAYS SHAM NTG-PLACEBO” group83.3% have IVb arrhythmias and 16.7% class IVa and in the “5 DAYS SHAMNTG” group 85.7% have IVb arrhythmias and 14.3% class IIIa arrhythmias.

FIG. 14: Arrhythmias during FPO (module 2: NO intermittent (NTG)).Numbers of columns are given in consecutive order of the columns ingroup IVb. Column 1 shows 5 DAYS RIP PBS; column 2 shows 5 DAYS RIPNTG-PLACEBO; column 3 shows 5 DAYS RIP NTG.

While arrhythmias in both control groups, PBS and NTG-Placebo, werepredominantly scaled into grade IVb, the NTG treated group was moreoften scaled into grade 0.

In the “5 DAYS RIP PBS” group, 75.0% of the rats have class IVbarrhythmias, 12.5% IVa and 12.5% class 0. Regarding the “5 DAYS RIPNTG-PLACEBO” group, 50.0% of the rats showed class IVb arrhythmias,16.7% IVa, 16.7% class IIIb and 16.7% class 0 arrhythmias.Interestingly, the “5 DAYS RIP NTG” group shows 42.9% class IVbarrhythmias and 57.1% class 0 arrhythmias.

FIG. 15: Arrhythmias during FPO (module 3: NO continuous (ISDN retard)).Numbers of columns are given in consecutive order of the columns ingroup IVb. Column 1 shows 5 DAYS RIP PBS; column 2 shows 5 DAYS RIPISDN-PLACEBO; column 3 shows 5 DAYS RIP ISDN.

In all groups, arrhythmias were similarly more often scaled into gradeIVb.

In the “5 DAYS ISDN-PLACEBO” group, 57.1% of the rats have class IVbarrhythmias, 14.3% class IVa and 28.6% class IIIb. The “5 DAYS RIP ISDN”group shows less severe arrhythmias with 57.1% class IVb, 28.6% classIIIb and 14.3% class 0 arrhythmias.

FIG. 16: Arrhythmias during FPO (module 4: NO intermittent plus ASA).Numbers of columns are given in consecutive order of the columns ingroup IVb. Column 1 shows 5 DAYS RIP ASA+PBS; column 2 shows 5 DAYS RIPASA+NTG-PLACEBO; column 3 shows 5 DAYS RIP ASA+NTG.

Arrhythmias were similarly scaled more into grade IVb in all groups.

In the “5 DAYS RIP ASA+PBS” group, in the group treated withASA+NTG-PLACEBO and in the “5 DAYS RIP ASA+NTG” group 83.3% of the ratsposses class IVb arrhythmias and 16.7% class IIIc.

FIG. 17: VPB-Score. Column 1 shows SHAM PBS; column 2 shows SHAMNTG-Placebo; column 3 shows SHAM NTG; column 4 shows RIP PBS; column 5shows RIP NTG-Placebo; column 6 shows RIP NTG; column 7 shows RIP PBS;column 8 shows RIP ISDN-Placebo column 9 shows RIP ISDN; column 10 showsRIP PBS+ASA; column 11 shows RIP NTG-Placebo+ASA; column 12 shows RIPNTG+ASA.

Regarding the percentage of each Lown grade of every group, a VBP scorecan be ascertained. The more animals show a higher grade, the higher isthe VBP score.

The VBP score shows the percentage of each Lown grade of every group.The Sham-groups have higher VBP-scores. Compared to the group with anischemic protocol (control group, treated with PBS), more rats showsevere arrhythmias. The treatment with NTG revealed reduced arrhythmias,and consequently a lower VPB-Score. The VPB-Score in groups treated withASA alone or NTG+ASA is higher compared to the controls (treated withPBS).

FIG. 18: Infarct size of 5-days- and 10-days-control-groups. Column 1shows 5 DAYS SHAM PBS, n=8: 13.36±5.22%; column 2 shows 5 DAYS RIP PBS,n=8: 11.05±5.12%; column 3 shows 10 DAYS SHAM PBS, n=7: 13.71±6.04%;column 4 shows 10 DAYS RIP PBS, n=6: 6.57±3.26%; standard deviation isindicated by error bars; asterisk indicates significant compared to theshams (nominal p-value<0.013).

After an ischemic protocol of 5 days there is no significantly smallerinfarct size measurable, but after a RIP of 10 days the infracted areais significantly decreased compared to the shams (nominalp-value<0.013).

After 90 minutes of LAD occlusion and 20 minutes reperfusion, infarctsize was analyzed.

The “10 DAYS RIP PBS” group has a significantly smaller infarct areacompared to the “10 DAYS SHAM PBS” group. There is no significancebetween both 5 DAYS groups.

FIG. 19: Infarct size (module 1: Sham Operation (without the RIP)).Column 1 shows 5 DAYS SHAM PBS, n=8: 13.36±5.22%; column 2 shows 5 DAYSSHAM NTG-PLACEBO, n=6: 14.21±5.79%; column 3 shows 5 DAYS SHAM NTG, n=7:14.09±5.18%; standard deviation is indicated by error bars.

The infarct size shows no difference between the SHAM groups.

There is no significance between the three SHAM groups.

FIG. 20: Infarct size (module 2: NO intermittent (NTG)). Column 1 shows5 DAYS RIP PBS, n=8: 11.05±5.12%; column 2 shows 5 DAYS NTG-PLACEBO:n=6; 9.80±6.79%; column 3 shows 5 DAYS RIP NTG, n=7: 3.61±2.08%;standard deviation is indicated by error bars, asterisk indicatessignificant compared to 5 DAYS RIP PBS (nominal p-value<0.017).

The infarct size is significantly smaller after treatment with NTGcompared to controls (treated with PBS) (nominal p-value<0.033).

Compared to the “5 DAYS RIP PBS”, a significantly smaller infarct areais observed in the “5 DAYS RIP NTG” group. There is no significancebetween the PBS and NTG-PLACEBO-group.

FIG. 21: Infarct size (module 3: NO continuous (ISDN retard)). Column 1shows 5 DAYS RIP PBS, n=8: 11.05±5.12%; column 2 shows 5 DAYSISDN-PLACEBO, n=6: 9.97±3.65%; column 3 shows 5 DAYS RIP ISDN, n=7:7.59±4.38%; standard deviation is indicated by error bars.

The infarct size after treatment with ISDN is smaller compared tocontrols (treated with PBS or ISDN-Placebo), but there is nosignificance.

The infarct size in the ISDN group (“5 DAYS RIP ISDN”) is smallercompared to the PBS group, as well as the ISDN-PLACEBO-group.

FIG. 22: Infarct size (module 4: NO intermittent plus ASA). Column 1shows 5 DAYS RIP PBS, n=8; 11.05±5.12%; column 2 shows 5 DAYS RIPASA+PBS, n=6: 12.51±3.05%; column 3 shows 5 DAYS NTG-PLACEBO: n=6;9.80±6.79%; column 4 shows 5 DAYS RIP NTG-PLACEBO+ASA, n=6: 13.92±1.71%;column 5 shows 5 DAYS RIP NTG, n=7: 3.61±2.08%; column 6 shows 5 DAYSRIP NTG+ASA, n=6: 13.00±3.82%; standard deviation is indicated by errorbars, asterisk indicates significant compared to 5 DAYS RIP NTG (nominalp-value<0.017).

The infarct size after treatment with NTG plus ASA is significantlyincreased compared to the treatment with NTG alone (nominalp-value<0.017).

The infarct size in the group treated with ASA (“5 DAYS ASA+PBS”) isminimally increased compared to the PBS control group, as well as theASA+NTG-PLACEBO-group. There is no difference between the ASA+NTG-groupand the group treated with ASS and PBS. However, the infarct area in theNTG group is significantly smaller compared to the ASA+NTG group.

FIG. 23: TTC-staining. The pictures show slices of three levels.Infarcted tissue stains a pale-white since they lack the enzymes withwhich the TTC reacts. Thus the areas of necrosis are clearly discernibleand quantifiable.

FIG. 24: Collateral diameters of ROI (module 1: Sham Operation (withoutthe RIP)). Column 1 shows 5 DAYS SHAM PBS, n=3: 82.7±3.7 μm; column 2shows 5 DAYS SHAM NTG-PLACEBO, n=3: 89.6 μm±10.6 μm; column 3 shows 5DAYS SHAM NTG, n=3: 86.8±9.0 μm; standard deviation is indicated byerror bars.

There is no growth of collaterals and no differences measurable betweenthe SHAM groups.

There is no significance between the three SHAM-groups.

FIG. 25: Collateral diameters of ROI (module 2: NO intermittent (NTG)).Column 1 shows 5 DAYS RIP PBS, n=3: 129.8±6.9 μm; column 2 shows 5 DAYSRIP NTG-PLACEBO: n=3; 127.0±12.1 μm; column 3 shows 5 DAYS RIP NTG, n=3:158.4±9.2 μm; standard deviation is indicated by error bars, asteriskindicates significant compared to 5 DAYS RIP NTG (nominalp-value<0.033). Diameters of collaterals are significantly increased bytreatment with NTG compared to controls (treated with PBS orNTG-Placebo) (nominal p-value<0.033).

Compared to the “5 DAYS RIP PBS”, the diameters of the collaterals inthe ROI in the “5 DAYS RIP NTG” group are significantly increased. Thereis no difference between the PBS and NTG-PLACEBO-group.

FIG. 26: Collateral diameters of ROI (module 3: NO continuous (ISDNretard)). Column 1 shows 5 DAYS RIP PBS, n=3: 129.8±6.9 μm; column 2shows 5 DAYS ISDN-PLACEBO, n=3: 133.0±11.5 μm; column 3 shows 5 DAYS RIPISDN, n=3: 148.2±11.3 μm; standard deviation is indicated by error bars.

No differences are measurable in the diameter of collaterals aftertreatment with ISDN or ISDN-Placebo.

The diameters of the collaterals in the ISDN group (“5 DAYS RIP ISDN”)are enhanced compared to the PBS group, as well as compared to theISDN-PLACEBO group.

FIG. 27: Collateral diameter of ROI (module 4: NO intermittent plusASA). Column 1 shows 5 DAYS RIP PBS, n=3; 129.8±6.9 μm; column 2 shows 5DAYS RIP PBS+ASA, n=3: 102.5±8.0 μm; column 3 shows 5 DAYS RIPNTG-PLACEBO: n=3; 127.0±12.1 μm; column 4 shows 5 DAYS NTG-PLACEBO+ASA,n=3: 97.1±8.6 μm; column 5 shows 5 DAYS RIP NTG, n=3: 158.4±9.2 μm;column 6 shows 5 DAYS RIP ASA+NTG, n=3: 124.4±5.6 μm; standard deviationis indicated by error bars, one asterisk indicates significant comparedto 5 DAYS RIP PBS (nominal p-value<0.039); double asterisk indicatessignificant compared to 5 DAYS RIP ASA+NTG (nominal p-value<0.039).

Diameters of collaterals are significantly smaller after treatment withASA compared to control (treated with PBS) (nominal p-value<0.039). Anadditional treatment with NTG abolished the inhibiting effect of ASA,but NTG-treatment alone shows significantly increased diameter comparedto treatment with NTG+ASA (**significant compared to 5 DAYS RIP ASA+NTG,nominal p-value<0.039).

The diameters in the group treated with PBS and ASA are significantlysmaller compared to the PBS control group, but there is no significancecompared to the ASA+NTG-PLACEBO-group. In the ASA+NTG-group diametersare significantly increased compared to the group treated with PBS andASA.

FIG. 28: MicroCT imaging of the “ROI”: (A) “5 DAYS SHAM PBS”; (B) “5DAYS SHAM NTG”; (C) “5 DAYS RIP ISDN”; (D) “5 DAYS RIP PBS”; (E) “5 DAYSRIP NTG”; (F) “5 DAYS RIP ASA+PBS; (G) “5 DAYS RIP ASA+NTG”.

The pictures show the growth of the collateral diameter in the region ofinterest by the ischemic protocol treated with PBS (D), NTG (E), or ISDN(C) compared to SHAM treated with PBS (A) or NTG (B). Inhibition ofcollateral growth by treatment with ASA (F) is partially abolished byadditional treatment with NTG (G).

EXAMPLES Example 1

450.2 g xylitol and 45.0 g tyloxapol are dissolved in 937.6 g ofpurified water. 67.8 g of a 5% solution of GTN in propylene glycol areadded and the mixture is stirred intensively for 15 minutes. A clearsolution is formed that is stable both at room temperature and at 4° C.The viscosity of this solution is 10 mPa*s.

It is concluded that a 0.225% solution of GTN can be prepared using thisformula.

Example 2

45.0 g xylitol and 3.0 g poloxamer 407 are dissolved in 46.0 g ofpurified water. 5.99 g of a 5% solution of GTN in propylene glycol areadded and the mixture is stirred intensively for 15 minutes. A clearsolution is formed that is stable both at room temperature and at 4° C.The viscosity of this solution is 17 mPa*s.

It is concluded that a 0.30% solution of GTN can be prepared using thisformula.

Example 3

35.5 g xylitol and 1.0 g poloxamer 407 are dissolved in 44.0 g ofpurified water. 5.01 g of a 5% solution of GTN in propylene glycol aredissolved in 15.0 g ethanol. Both solutions are mixed and the mixture isstirred intensively for 15 minutes. A clear solution is formed that isstable both at room temperature and at 4° C. The viscosity of thissolution is 12 mPa*s.

It is concluded that a 0.25% solution of GTN can be prepared using thisformula.

Example 4

35.1 g xylitol and 1.0 g tyloxapol are dissolved in 44.0 g of purifiedwater. 5.06 g of a 5% solution of GTN in propylene glycol are dissolvedin 15.0 g ethanol. Both solutions are mixed and the mixture is stirredintensively for 15 minutes. A clear solution is formed that is stableboth at room temperature and at 4° C. The viscosity of this solution is10 mPa*s.

It is concluded that a 0.25% solution of GTN can be prepared using thisformula.

Example 5

1125.0 g xylitol and 125.1 g poloxamer 407 are dissolved in 1100 g ofpurified water. 150.0 g of a 5% solution of GTN in propylene glycol areadded and the mixture is stirred intensively for 15 minutes. The pHvalue of the solution is adjusted to pH 4.5 with lactic acid. A clearsolution is formed that is stable both at room temperature and at 4° C.Furthermore this solution can be frozen at −20° C. and results in aclear solution after thawing. The viscosity of this solution is 25mPa*s.

It is concluded that a 0.30% solution of GTN can be prepared using thisformula. The long term stability of this solution is excellent as can beseen from the following table:

Stability at 40° C. Start 3 months 6 months GTN assay (HPLC) 0.296%0.293% 0.287% Stability at 25° C. Start 6 months 12 months GTN assay(HPLC) 0.296% 0.293% 0.294%

Example 9

30.0 g poloxamer 407 are dissolved in 1169 g of purified water. 75.8 gof a 5 solution of GTN in propylene glycol and 225 g ethanol are addedand the mixture is stirred intensively for 15 minutes. The pH value ofthe solution is adjusted to pH 4.5 with lactic acid. A clear solution isformed that is stable at room temperature and at 4° C. Furthermore thissolution can be frozen at −20° C. and results in a clear solution afterthawing.

It is concluded that a 0.25% solution of GTN can be prepared using thisformula. The long term stability of this solution is excellent as can beseen from the following table:

Stability at 40° C. Start 3 months 6 months GTN assay (HPLC) 0.252%0.252% 0.245% Stability at 25° C. Start 6 months GTN assay (HPLC) 0.252%0.246%

Example 10

5.5 g tyloxapol are dissolved in 84.4 g of purified water. 10.0 g of a5% solution of GTN in propylene glycol and 0.10 g benzoic acid are addedand the mixture is stirred intensively for 15 minutes. The pH value ofthe solution is adjusted to pH 4.1 with lactic acid. A clear solution isformed that is stable at room temperature and at 4° C. It is concludedthat a 0.5% solution of GTN can be prepared using this formula.

Example 11

0.10 g benzoic acid, 6.0 g tyloxapol and 3.0 g poloxamer are dissolvedin 50.8 g of purified water. 30.0 g xylitol are added and dissolved bystirring. 10.0 g of a 5 solution of GTN in propylene glycol are addedand the mixture is stirred intensively for 15 minutes. The pH value ofthe solution is adjusted to pH 4.0 with lactic acid. A clear solution isformed that is stable at room temperature and at 4° C. It is concludedthat a 0.5% solution of GTN can be prepared using this formula.

Example 12

450.2 g xylitol, 45.0 g tyloxapol and 3.76 g sodium benzoate aredissolved in 929.6 g of purified water. 67.8 g of a 5% solution of GTNin propylene glycol are added and the mixture is stirred intensively for15 minutes. The pH value is adjusted to pH 4.0 with lactic acid. A clearsolution is formed that is stable both at room temperature and at 4° C.The viscosity of this solution is 10 mPa*s.

It is concluded that a 0.22% solution of GTN can be prepared using thisformula.

The long term stability of this solution is excellent as can be seenfrom the following table:

Stability at 40° C. Start 3 months 6 months GTN assay (HPLC) 0.219%0.220% 0.221% Stability at 25° C. Start 3 months 12 months GTN assay(HPLC) 0.219% 0.222% 0.228%

Example 13

10.1 g tyloxapol are dissolved in 158.0 g of purified water. 20.0 g of a5% solution of GTN in ethanol and 11.6 g ethanol are added and mixed.The pH value is adjusted to pH 4.0 with lactic acid. The mixture isstirred intensively for 15 minutes. A clear solution is formed that isstable both at room temperature and at 4° C.

It is concluded that a 0.50% solution of GTN can be prepared using thisformula.

Comparative Example 1

30.0 g xylitol are dissolved in 65.5 g of purified water. 4.51 g of a 5%solution of GTN in propylene glycol are added and the mixture is stirredintensively for 15 minutes. Immediately after production droplets of GTNare observed at the bottom of the glass beaker (FIG. 1).

It is concluded that a solution of 0.225% of GTN can not be made in thisway.

Comparative Example 2

30.7 g xylitol and 1.0 g tyloxapol are dissolved in 64.5 g of purifiedwater. 4.52 g of a 5% solution of GTN in propylene glycol are added andthe mixture is stirred intensively for 15 minutes. Immediately afterproduction the solution is turbid. After three days of storage at roomtemperature a turbid phase at the bottom of the glass vial and a clearphase above are observed.

It is concluded that 1% tyloxapol is not sufficient to dissolve 0.225%of GTN in this formula.

Comparative Example 3

45.0 g xylitol are dissolved in 49.0 g of purified water. 6.03 g of a 5%solution of GTN in propylene glycol are added and the mixture is stirredintensively for 15 minutes. Immediately after production droplets of GTNare observed at the bottom of the glass beaker (FIG. 2).

It is concluded that a solution of 0.30% of GTN can not be made in thisway.

Comparative Example 4

45.1 g xylitol and 1.5 g poloxamer 407 are dissolved in 47.5 g ofpurified water. 6.01 g of a 5% solution of GTN in propylene glycol areadded and the mixture is stirred intensively for 15 minutes. Immediatelyafter production the solution is slightly turbid. After three days ofstorage at room temperature a turbid phase at the bottom of the glassvial and an opalescent phase above are observed.

It is concluded that 1.5% poloxamer is not sufficient to dissolve 0.30%of GTN in this formula.

Comparative Example 5

35.0 g xylitol are dissolved in 45.0 g of purified water. 5.00 g of a 5%solution of GTN in propylene glycol are dissolved in 15.0 g ethanol.Both solutions are mixed and the mixture is stirred intensively for 15minutes. Immediately after production the mixture is very turbid, butvisually homogeneous. After 3 days of storage at 5° C. droplets of GTNseparated from the solution and are observed at the bottom of the glassvial.

Surprisingly the 60-fold excess of ethanol as compared to the amount ofGTN is not sufficient to yield a stable solution. It is concluded that asolution of 0.25% of GTN can not be made in this way.

The examples clearly demonstrate that a homogeneous solution is onlyobtained in the presence of the water soluble polymer. This is alsotrue, if ethanol is used as a preservative. Even if ethanol is presentin a concentration of 20 weight percent, this amount of ethanol is notsufficient to solubilise the GTN without a water soluble polymer. Forthe patient's safety it is required that no phase separation oraccumulation of the active substance occurs during storage at lowertemperatures (e.g. outside of the house in winter). GTN has a tendencyto accumulate at the bottom of the bottle. The dip tube of a spraydevice would suck this part of the solution first and a patient could beseriously overdosed in this case.

Example 6

This example has been taken from PCT/EP2013/061131 and is includedherein to demonstrate the pro-arteriogenic capacities of GTN. Since GTNis the active component of the pharmaceutical composition of theinvention, it is concluded that the pharmaceutical composition of theinvention is also capable of inducing arteriogenesis.

Pre-Clinical Study

1. Introduction

One important mechanism of arteriogenesis is the induction of shearstress across recruited collateral arteries.

NO plays a fundamental role in this scenario, since it regulates thevasodilatory capability of the artery as well as therapeuticproliferation aspects on the smooth muscle cells of collateral arteries.

Here we evaluated the effects of Nitrolingual Akut® Spray (G.Pohl-Boskamp GmbH & Co.KG, Hohenlockstedt, Germany; U.S. American brandname Nitrolingual® Pumpspray) in a unique non-myocardial infarctarteriogenesis model. Collateral growth in this model is induced viarepetitive occlusion of the left anterior descending coronary artery(LAD). Infarct size in these animals was measured as the endpoint at theend of the experiment. Thus, no interference between myocardialinfarction and arteriogenesis has weaken the experiment. Moreover weevaluated the effect of acetyl salicylic acid (ASA) in this model ofrepetitive coronary occlusion as a possible inhibitor of arteriogenesis.We evaluated whether a concomitant application of NO (intermittent useof nitroglycerin) may compensate for this negative effect of ASA.

2. Materials and Methods

1.1. Animal Preparation

Male Sprague-Dawley rats (300 g body weight at study start; n=182) areused for experiments. For surgery (day 0), rats are premedicated(ketamine 50 mg/ml plus xylazine 4 mg/ml intraperitoneal) and intubated.Oral intubation (I4-G polyethylene tubing) is done under directobservation of the vocal cords with an otoscope. General anesthesia isintroduced and maintained by isoflurane inhalation (1.0% to 2.0%, with100% oxygen). Body temperature is controlled at 37° C. by an electricheating table. Surgery is performed using aseptic technique. The animalis initially placed on its dorsal side and cutaneous clips are fixed.With a BioAmp differential amplifier coupled to a PowerLab dataacquisition system (AD Instruments) ECG parameters (heart rate) aremonitored and recorded during surgery. The heart is exposed by leftthoracotomy. A mini-pneumatic snare occluder (see the Mini-PneumaticSnare Occluder section for details) is implanted around the mid toproximal left anterior descending coronary artery (LAD). Confirmationthat the occluder is functional, i.e., producing myocardial ischemia, isdetermined initially by observation of blanching and hypokinesis of theleft ventricle (LV) and by observation of the electrocardiogram (STelevation) during inflation. Rats are randomly divided into 4therapeutic modules:

-   -   Module 1: Sham Operation    -   Module 2: NO intermittent (nitroglycerin)    -   Module 3: NO continuous (retard preparation of isosorbide        dinitrate)    -   Module 4: NO intermittent plus ASA

After instrumentation and measurements, the chest is closed underpositive end-expiratory pressure, and the thoracic cavity is evacuatedof air. The occluders are tunneled subcutaneously and exteriorizedbetween the scapulae. These catheters are protected by a stainless steelspring coil connected to a ring that is secured subcutaneously betweenthe scapulae. After the surgery, analgesic (buprenorphine 0.05 mg/kg SC)and antibiotic (enrofloxacin 10 mg/kg SC) are administered. Rats areobserved in a recovery cage for 2 hours and then transferred to theanimal care facility where they are continuously monitored bytechnicians. Postoperatively, buprenorphine (0.5 mg/kg SC) is given forpain twice a day for 8 resp. 13 days. On the third day after the surgery(day 3), ischemic protocol is started. After 5 resp. 10 days (only inmodule 1A and 2A) of the experimental protocol (day 8 resp. day 13), therats are anesthetized, and the chest is opened by mid thoracotomy. Inthe micro-CT group, the hearts are immediately excised. For the finalinfarct size detection the LAD is permanently occluded (final permanentocclusion, FPO) and infarct size is measured via TTC staining.

1.2. Mini-Pneumatic Snare Occluder for Rat Heart

A mini-pneumatic snare occluder is used consisting of a mini-balloon,sheath tubing, suture, and catheter. The balloon (7 mm long) is made ofsoft latex membrane and is sufficiently pliable to give negligiblephysical force on the coronary vessels during balloon deflation. Theballoon is mounted within an umbrella sheath (3.2 or 4.8 mm in diameter,12 mm in length; protects the balloon from fibrous infiltration).Prolene (5-0) is passed around the LAD and attached to the sheath,securing the occluder to the heart, so that myocardial ischemia isproduced by balloon inflation. Inflation volume is small (0.2 to 0.25 mLair), but occlusion occurs by 2 physical actions: “crimping” the LADtoward upward/outside and compressing the LAD by the inflatedballoon/sheath. The balloon is connected to a catheter (PE-50) that isexteriorized. Balloon inflation and deflation are controlled fromoutside the rat cage.

1.3. Measurements of ECG Parameters

In all four modules (1-4) at the beginning (day 3) and the end (day 8resp. day 13) of the experimental protocol (RIP) the coronary occlusionis performed for 40 seconds (equivalent to an occlusion in the RIP; seepage 6) and during FPO for 90 minutes (day 8 resp. day 13) ECGparameters are measured to examine the heart rate and ST elevation.Furthermore, the occurring arrhythmias during FPO are determined.According to Lown's classification, every animal shows a certain grade.The higher a grade, the more severe arrhythmias are. To illustrate themean severity of an entire group more descriptive, a VPB score isascertained. For that, every Lown grade refers to a particular factor(grade 0=factor 0; grade I=factor 1; grade II=factor 2; gradeIIIa=factor 3; grade IIIb=factor 4; grade VIa=factor 5; grade VIb=factor6 and grade V=factor 7). Every group has a different percentage ofanimals presenting each grade. The percentage of the respective gradesare multiplied with the appropriate factor leading to individual resultswhich are then summed up to the VPB score of the whole group.Consequently, a group of animals with higher Lown grades has acorrelatively high VPB score.

1.4. Coronary Microvascular Imaging with Micro-CT

In addition Micro-CT is used as a further endpoint to image collaterals.One group of rats (3 rats of each group in each module; total of 36rats) is prepared for coronary vascular visualization via micro-CT. Thecoronary circulation is filled with contrast medium (yellow microfil) bymodification of the methodology for micro-CT study in the rats. Theviscosity of the contrast medium enables filling up to coronaryarteriolar level with no or minimal filling of capillaries. The excisedheart is immediately cannulated by an aortic cannula, and coronarycirculation is perfused retrogradely at 85 mm Hg. A perfusate (25° C. to27° C. saline with 2% procaine) is used to avoid myocardial metaboliccontraction and maximally dilate the coronary vasculature. Polyethylenetubing is inserted into the LV via a left appendage through the mitralvalve to unload the LV. Warmed contrast medium (42° C.) is injected at apressure of 85 mmHg for 3 minutes while perfusion pressure is monitored.The heart is cooled by immersion into cold saline (0° C. to 4° C.) untilthe (yellow microfil) solidified. Then, the heart is removed and fixedin 4% paraformaldehyde solution (4° C.) overnight. Whole hearts are usedfor micro-CT imaging of coronary collateral growth. The coronaryvasculature is visualized with micro-CT. In brief, the whole heart isscanned in 1° increments around 360° about its apex-to-base longitudinalaxis. The spatial resolution selected in the present study has an18*18*18 m3 voxel size to focus on the size of collateral vessels and tominimize the signals from smaller vessels. Finally, CT data arereconstructed as 3D images. The main purpose of these images is toestablish the presence or absence of arterial-arterial anastomoticconnections. Collateral vessels, i.e., arterial-arterial anastomoticconnections, are measured by independent observers for the groups.Collateral arterial network morphology is analyzed with Amira 5.2.2software (Visage Imaging, Berlin, Germany).

1.5. Experimental Protocol

The repetitive ischemia protocol (RIP) is introduced by automatisedinflation of the occluder using the following protocol: 40 seconds ofocclusion every 20 minutes for 2 hours 20 minutes, followed by a periodof “rest” (deflation) for 5 hours 40 minutes. This 8-hour set isrepeated 3 times a day for 5 resp. 10 days (only in module 1A and 2A).The LAD is occluded automatically by remote inflation or deflationthrough the catheter. In sham rats (see module 1), the balloon isimplanted, but RIP is not applied. Rats under RI protocol are randomlydivided into the three modules 2, 3 and 4.

1.6. Infarct Size Detection

Infarct size is detected by TTC staining after final permanentocclusion. After 5 resp.

10 days (only in module 1A and 2A) of the experimental protocol, theoccluder is inflated permanently for 90 minutes. Infarct size ismeasured by TTC staining (n=10/group). Therefore rats are anaesthesizedand undergo again the ECG recording to confirm the occlusion (STelevation) and to calculate ECG parameters and the numbers ofarrhythmias. In animals without collaterals, coronary occlusion causesdeterioration of systemic hemodynamics and arrhythmias, includingpremature ventricular contractions, ventricular tachycardia, andventricular fibrillation; in animals with well developed collaterals, nosuch adverse effects are noted. The ECG parameters were recorded andanalysed using a computerized program (Lab chart 7).

The chest is opened by mid thoracotomy. The heart is immediately excisedand sectioned from apex to base in 2-mm-thick transverse slices parallelto the atrioventricular groove. Slices are incubated with 0.09 mol/Lsodium phosphate buffer containing 1.0% triphenyl tetrazolium chloride(TTC) and 8% dextran for 20 min. at 37° C. Slices are fixed in 10%formaldehyde and then photographed with a digital camera mounted on astereomicroscope. The infarcted size is quantified using a computerizedplanmetric program (Adobe Photoshop). The infarcted area is indentifiedas the TTC-negative tissue and is expressed as a percentage of the areaof the left ventricle (LV).

1.7. Details Regarding Testing Compounds

-   ASA Merck Chemicals-   NO intermittent (NTG) nitroglycerin solution; Nitrolingual Akut®    Spray, G. Pohl-Boskamp GmbH & Co. KG, Hohenlockstedt, Germany-   NO continuous (ISDN retard) isosorbide dinitrate retard pellets;    Nitrosorbon® retard; G. Pohl-Boskamp GmbH & Co. KG, Hohenlockstedt,    Germany    Carrier Compound for NO Intermittent-   (NTG-Placebo) placebo solution of Nitrolingual Akut® Spray,    Pohl-Boskamp GmbH & Co. KG, Hohenlockstedt, Germany    NO Continuous Carrier Compound-   (ISDN-Placebo) neutral pellets of Nitrosorbon® retard; G.    Pohl-Boskamp GmbH & Co. KG, Hohenlockstedt, Germany-   Control buffer PBS (phosphate buffered saline)

1.8. Route, Timepoint and Concentration of Delivery to Animals

All medication (ASA and NTG and ISDN retard) is given upfront to afollowing occlusion time of the device. The control buffer (PBS) isgiven in the same way prior to the first two occlusions.

NO Intermittent (NTG)

A new test solution is prepared every morning at eight o'clock. Thesolution is taken from the vials via syringes.

NO intermittent (NTG) is given twice a day with a time interval of 8hours.

Due to the chronic instrumentation of the rats and to avoid furtherstress, NTG is given via buccal application. 50 μl of the daily preparedtest solution containing 17.37 μg nitroglycerin (equivalent to a humandose of 0.8 mg, as calculated by the formula dosis/animal [mg]=metabolicbody weight [kg0,75]*human dosis [mg/kg]*recalculation factor[kg/kg0,75] according to Loscher, W., Ungemach, F.R., Kroker, R., 1998,Blackwell Science, 3rd edition) is administered per buccal applicationin module 1, 2 and 4. The time point of application is directly upfrontto balloon inflation at 9 a.m. and 5 p.m., thus with maximal effects onrecruited collateral arteries.

This concentration is taken from the above mentioned reaction vialsright before administration.

Carrier compound solution served as a stock solution for the preparationof the test solution.

Carrier Compound for NO Intermittent (NTG-Placebo)

Carrier compound is administered in a way identical to NO intermittent.

NO Continuous (ISDN Retard)

The medication for prolonged NO delivery (retard preparation isosorbidedinitrate=long-acting nitrate ISDN) is delivered as retarded pellets 1×per day.

For the retard preparation ISDN in a dosage of 2.6 mg ISDN/rat ischosen. Therefore 13 mg pellets are suspended in 0.5 ml drinking waterand are applied via gavage at 9 a.m. every morning (equivalent of ahuman dose of 2 mg/kg/bw).

NO Continuous Carrier Compound (ISDN-Placebo)

Carrier compound is administered in a way identical to NO continuous.

No Intermittent Plus ASA (Acetylsalicylic Acid)

Every morning at 9.30 a.m. 2.22 mg ASA per rat is given dissolved in 0.5ml drinking water via gavage directly into the stomach.

The ASA concentration of 2.22 mg ASA per rat (6.34 mg/kg bw) correlateswith the human dosage of 100 mg/day.

1.9. Animals and Groups

10 rats per groups (FPO=final permanent occlusion to induce infarcts)Group d: 3 additional animals are treated with the same medications andligation scheme like the corresponding groups a, b and c, but withoutFPO. These 9 animals per module are used for micro CT images.

Module 1: Sham Operation (without the RIP):

-   A. Control buffer (phosphate buffered saline PBS) with functional    FPO for infarct size detection n=20    -   1. n=10: “5 DAYS SHAM PBS”    -   2. n=10 “10 DAYS SHAM PBS”-   B. Carrier compound without NO plus functional FPO for infarct size    detection    -   n=10: “5 DAYS SHAM NTG-PLACEBO”-   C. NTG with functional FPO for infarct size detection    -   n=10: “5 DAYS SHAM NTG”-   D. A1.) n=3 A2.) n=3 B) n=3 C) n=3 for micro CT images    -   n=12    -   total: n=52        Module 2: NO Intermittent:-   A. intermittent control buffer with functional FPO for infarct size    detection    -   n=20    -   1. n=10: “5 DAYS RIP PBS”    -   2. n=10: “10 DAYS RIP PBS”-   B. intermittent Carrier compound plus functional FPO for infarct    size detection    -   n=10: “5 DAYS RIP NTG-PLACEBO”-   C. Intermittent NTG with functional FPO for infarct size detection    -   n=10: “5 DAYS RIP NTG”-   D. A1.) n=3 A2.) n=3 B) n=3 C) n=3 for micro CT images    -   n=12    -   total: n=52        Module 3: NO Continuous:-   A. Continuous Control buffer (drinking water) with functional FPO    for infarct size detection (n=10): “5 DAYS RIP DW”-   B. Continuous Carrier compound plus functional FPO for infarct size    detection n=10: “5 DAYS RIP ISDN-PLACEBO”-   C. Continuous NO functional FPO for infarct size detection    -   n=10: “5 DAYS RIP ISDN”-   D. A.) n=3 B.) n=3 C.) n=3 for micro CT images    -   n=9    -   total: n=(39)        Module 4: NO Intermittent Plus ASA:-   A. Intermittent Control buffer plus ASA with functional FPO for    infarct size detection n=10: “5 DAYS RIP PBS+ASA”-   B. Intermittent NO Carrier compound plus ASA plus functional FPO for    infarct size detection n=10: “5 DAYS RIP NTG-PLACEBO+ASA”-   C. Intermittent NTG plus ASA functional FPO for infarct size    detection    -   n=10: “5 DAYS RIP NTG+ASA”-   D. A.) n=3 B.) n=3 C.) n=3 for micro CT images    -   n=9    -   total: n=39

2. Statistical Analysis

All data are given as mean±SD. Graphics are shown as mean±SEM.

Results obtained by measuring ST segment elevation, infarct size andvessel diameters are analysed for statistical significance by using theSPSS 20 software package (IBM SPSS Statistics, NY, USA). ANOVA with afalse discovery rate, FDR, correction is used. p values are adjusted formultiple testing using a FDR procedure to achieve an experiment-widesignificance of p 0.05. FDR takes into account the number of nullhypotheses rejected and has been shown to increase statistical power ascompared to Bonferroni correction.

3. Results

3.1 Final Permanent Occlusion

LAD occlusion allowed a prospective study of the function of collateralvessels. Such vessels can protect myocardial tissue at risk of ischemiaafter coronary occlusion.

At the end of the RI protocol the permanent LAD occlusion is performedin one subgroup of all groups and ECG parameters to examine ST segmentelevation and ventricular arrhythmias are measured. After 90 minutes ofpermanent occlusion the infarcted area is determined.

3.2 ECG Analysis

Electrocardiographic manifestations of ischemia initiated by LADocclusion are less pronounced when collateral vessels are present.

3.3 ST Segment Elevation

During LAD occlusion there is an inverse correlation between themagnitude of ST segment elevation and the extent of the collateralsupply.

Collateral function is an important determinant of the direction of STsegment response to ischemia during acute coronary occlusion. ReversibleST segment elevation during acute LAD occlusion is related to inadequatecollateral arterial function. In patients with reversible ST segmentdepression, coronary collateral function appears to be better and, as aconsequence, shows less ischemia results.

During the 90 minutes occlusion the ST segment elevation in the “10 DAYSSHAM PBS” is significantly higher compared to the “10 DAYS RIP PBS”group (10 DAYS SHAM, n=7: 0.124±0.039 mV; 10 DAYS RIP, n=7: 0.055±0.033mV). In contrast, ST segment elevation in the “5 DAYS SHAM PBS” issimilar to the “5 DAYS RIP PBS” group (5 DAYS SHAM, n=8: 0.134±0.034 mV;5 DAYS RIP, n=8: 0.104±0.016 mV) (FIGS. 3 and 4).

Module 1: Sham Operation (without the RIP)

There is no significance between the three SHAM-groups (5 DAYS SHAM PBS,n=8: 0.134±0.034 mV; 5 DAYS SHAM NTG-PLACEBO, n=6: 0.131±0.043 mV; 5DAYS SHAM NTG, n=7: 0.124±0.058 mV) (FIGS. 5 and 6).

Module 2: NO Intermittent (NTG)

In the NTG group (“5 DAYS RIP NTG”) ST elevation is significantlydecreased compared to the PBS group (5 DAYS RIP PBS, n=8: 0.104±0.016mV; 5 DAYS RIP NTG, n=7: 0.052±0.030 mV). There is no significancebetween the PBS and NTG-PLACEBO-group (5 DAYS NTG-PLACEBO: n=6;0.096±0.061 mV) (FIGS. 7 and 8).

Module 3: NO Continuous (ISDN Retard)

ST segment elevation in the ISDN group (“5 DAYS RIP ISDN”) is decreasedcompared to the PBS group (5 DAYS RIP PBS, n=8: 0.104±0.016 mV; 5 DAYSRIP ISDN, n=7: 0.062±0.027 mV), but there is no significance as well asbetween the PBS and ISDN-PLACEBO-group (5 DAYS ISDN-PLACEBO, n=7:0.110±0.069 mV) (FIGS. 9 and 10).

Module 4: NO Intermittent Plus ASA

ST segment elevation in the group treated with PBS and ASA is highercompared to the PBS control group (5 DAYS RIP ASA+PBS, n=7: 0.138±0.098mV; 5 DAYS RIP PBS, n=8; 0.104±0.016 mV), but there is no significanceas well as between the ASA+NTG-PLACEBO-group (5 DAYS RIPASA+NTG-PLACEBO, n=6: 0.144±0.091 mV). In the ASA+NTG-group ST elevationis decreased compared to the group treated with ASA and PBS (5 DAYS RIPNTG+ASA, n=7: 0.088±0.071 mV) (FIGS. 11 and 12).

3.4. Ventricular Arrhythmias

The importance of ventricular premature beats (VPBs) results from theirpossible association with an increased risk for cardiac sudden death.VPBs were stratified according to the Lown classification. A high Lowngrade has been shown to predict mortality after acute myocardialinfarction.

-   Grade 0: no ventricular ectopic beats-   Grade I: occasional, isolated VPB-   Grade II: frequent VPB (>1/min or 30/h)-   Grade III: multiform VPB    -   (a) VPB    -   (b) Bigenimus-   Grade IV: repetitive VPB    -   (a) Couplets    -   (b) Salvos-   Grade V: Early VPB    Module 1: Sham Operation (without the RIP)

In the “5 DAYS SHAM PBS” group 87.5% of the rats have class IVbarrhythmias and 12.5% class IVa. In the “5 DAYS SHAM NTG-PLACEBO” group83.3% have IVb arrhythmias and 16.7% class IVa and in the “5 DAYS SHAMNTG” group 85.7% have IVb arrhythmias and 14.3% class IIIa arrhythmias(FIG. 13).

Module 2: NO Intermittent (NTG)

In the “5 DAYS RIP PBS” group, 75.0% of the rats have class IVbarrhythmias, 12.5% IVa and 12.5% class 0. Regarding the “5 DAYS RIPNTG-PLACEBO” group, 50.0% of the rats showed class IVb arrhythmias,16.7% IVa, 16.7% class IIIb and 16.7% class 0 arrhythmias.Interestingly, the “5 DAYS RIP NTG” group shows 42.9% class IVbarrhythmias and 57.1% class 0 arrhythmias (FIG. 14).

Module 3: NO Continuous (ISDN Retard)

In the “5 DAYS ISDN-PLACEBO” group, 57.1% of the rats have class IVbarrhythmias, 14.3% class IVa and 28.6% class IIIb. The “5 DAYS RIP ISDN”group shows less severe arrhythmias with 57.1% class IVb, 28.6% classIIIb and 14.3% class 0 arrhythmias (FIG. 15).

Module 4: NO Intermittent Plus ASA

In the “5 DAYS RIP ASA+PBS” group, in the group treated withASS+NTG-PLACEBO and in the “5 DAYS RIP ASS+NTG” group 83.3% of the ratsposses class IVb arrhythmias and 16.7% class IIIa (FIG. 16).

Regarding the percentage of each Lown grade of every group, a VBP scorecan be ascertained. The more animals show a higher grade, the higher isthe VBP score (FIG. 17).

FIG. 19: VPB-Score

TABLE 1 VPB-Score group VPB-Score Module 1 SHAM PBS 5.88 SHAMNTG-PLACEBO 5.83 SHAM NTG 5.71 Module 2 RIP PBS 5.13 RIP NTG-PLACEBO4.50 RIP NTG 2.57 Module 3 RIP PBS 5.13 RIP ISDN-PLACEBO 5.29 RIP ISDN4.57 Module 4 RIP ASA + PBS 5.50 RIP ASA + NTG-PLACEBO 5.50 RIP ASA +NTG 5.50

3.5. Infarct Size

After 90 minutes of LAD occlusion and 20 minutes reperfusion, infarctsize was analyzed.

The “10 DAYS RIP PBS” group has a significantly smaller infarct areacompared to the “10 DAYS SHAM PBS” group (10 DAYS RIP PBS, n=6:6.57±3.26%; 10 DAYS SHAM PBS, n=7: 13.71±6.04%). There is nosignificance between both 5 DAYS groups (5 DAYS SHAM PBS, n=8:13.36±5.22%; 5 DAYS RIP PBS, n=8: 11.05±5.12%) (FIG. 18).

Module 1: Sham Operation (without the RIP)

There is no significance between the three SHAM-groups (5 DAYS SHAM PBS,n=8: 13.36±5.22%; 5 DAYS SHAM NTG-PLACEBO, n=6: 14.21±5.79%; 5 DAYS SHAMNTG, n=7: 14.09±5.18%) (FIG. 19).

Module 2: NO Intermittent (NTG)

Compared to the “5 DAYS RIP PBS”, a significantly smaller infarct areais observed in the “5 DAYS RIP NTG” group (5 DAYS RIP PBS, n=8:11.05±5.12%; 5 DAYS RIP NTG, n=7: 3.61±2.08%). There is no significancebetween the PBS and NTG-PLACEBO-group (5 DAYS NTG-PLACEBO: n=6;9.80±6.79%) (FIG. 20).

Module 3: NO Continuous (ISDN Retard)

The infarct size in the ISDN group (“5 DAYS RIP ISDN”) is smallercompared to the PBS group (5 DAYS RIP PBS, n=8: 11.05±5.12%; 5 DAYS RIPISDN, n=7: 7.59±4.38%), as well as the ISDN-PLACEBO-group (5 DAYSISDN-PLACEBO, n=6: 9.97±3.65%) (FIG. 21).

Module 4: NO Intermittent Plus ASA

The infarct size in the group treated with ASA (“5 DAYS ASA+PBS”) isminimally increased compared to the PBS control group (5 DAYS RIPASA+PBS, n=6: 12.51±3.05%; 5 DAYS RIP PBS, n=8; 11.05±5.12%), as well asthe ASA+NTG-PLACEBO-group (5 DAYS RIP ASA+NTG-PLACEBO, n=6:13.92±1.71%). There is no difference between the ASA+NTG-group and thegroup treated with ASA and PBS (FIG. 22). However, the infarct area inthe NTG group is significantly smaller compared to the ASA+NTG group (5DAYS RIP NTG, n=7: 3.61±2.08%; 5 DAYS RIP NTG+ASS, n=6: 13.00±3.82%)(FIG. 23).

3.6. Coronary Microvascular Imaging with Micro-CT

Collateral arteries are pre-existent vessels running parallel to a majorartery. In case the major artery is occluded, even for a short period oftime (40 sec during this RIP), collaterals assume the blood supply. As aresult, collateral arteries in this area (ROI, region of interest) startto grow in length (clearly visible by the cork screw pattern) and mostnotably in their diameter. So we measured the diameter of thecollaterals in the ROI.

Module 1: Sham Operation (without the RIP)

There is no significance between the three SHAM-groups (5 DAYS SHAM PBS,n=3: 82.7±3.7 μm; 5 DAYS SHAM NTG-PLACEBO, n=3: 89.6 μm±10.6 μm; 5 DAYSSHAM NTG, n=3: 86.8±9.0 μm) (FIGS. 24 and 28).

Module 2: NO Intermittent (NTG)

Compared to the “5 DAYS RIP PBS”, the diameters of the collaterals inthe ROI in the “5 DAYS RIP NTG” group are significantly increased (5DAYS RIP PBS, n=3: 129.8±6.9 μm; 5 DAYS RIP NTG, n=3: 158.4±9.2 μm).There is no difference between the PBS and NTG-PLACEBO-group (5 DAYSNTG-PLACEBO: n=3; 127.0±12.1 μm) (FIGS. 25 and 28).

Module 3: NO Continuous (ISDN Retard)

The diameter of the collaterals in the ISDN group (“5 DAYS RIP ISDN”)are enhanced compared to the PBS group (5 DAYS RIP PBS, n=3: 129.8±6.9μm; 5 DAYS RIP ISDN, n=3: 148.2±11.3 μm), as well as compared to theISDN-PLACEBO group (5 DAYS ISDN-PLACEBO, n=3: 133.0±11.5 μm) (FIGS. 26and 28).

Module 4: NO Intermittent Plus ASA

The diameter in the group treated with PBS and ASA are significantlysmaller compared to the PBS control group (5 DAYS RIP PBS+ASA, n=3:102.5±8.0 μm; 5 DAYS RIP PBS, n=3; 129.8±6.9 μm), but there is nosignificance compared to the ASA+NTG-PLACEBO-group (5 DAYSNTG-PLACEBO+ASA, n=3: 97.1±8.6 μm). In the ASA+NTG-group diameter aresignificantly increased compared to the group treated with PBS and ASA(5 DAYS RIP ASA+NTG, n=3: 124.4±5.6 μm) (FIGS. 27 and 28).

4. Conclusion

We examined the groups “10 DAYS SHAM PBS” and “5 DAYS SHAM PBS”, eachwithout a RIP (repetitive ischemic protocol) and the groups “10 DAYS RIPPBS” and “5 DAYS RIP PBS”, each with a RIP of five and ten days.

Measurement of infarct volume after a 90 minute permanent LAD occlusion(FPO, final permanent occlusion) revealed significantly smallerinfarcted areas in the 10 DAYS RIP group than in “10 DAYS SHAM” group.In contrast, after a RIP of five days, no differences became apparent inthe SHAM and RIP group.

Moreover, we used ECG parameters for examinations and evaluation for thefirst time. We found the maximal ST elevation after FPO of the LADshowed no crucial differences between “5 DAYS RIP PBS” and SHAM groups,yet. However, after 10 days ST elevations were significantly decreasedin the RIP group.

Aside from ST elevation measurement during FPO, we were able to analyzeand evaluate arrhythmias in differentiated way.

Based on these novel insights into the characterization of rat RMImodel, we decided to use a 5 day RIP in case of an expected stimulationof arteriogenesis.

The degree of ST elevation enhancement and the infarct volume after a 10day RIP can be obtained with pro-arteriogenic substances within a 5 dayRIP, yet.

This provides additional parameters being able to approve our results ofinfarct volume measurement.

The intermittent application of NTG solution (twice daily on buccalmucosa) decreased serious arrhythmias of the rat heart during FPOcompared to the control group. Additionally, infarct volume is decreasedby more than 50% after 90 minutes FPO compared to the control group.This reduction in infarct size is not even obtainable with controls setto a 10 days RIP. Furthermore, a treatment with NTG solutionsignificantly attenuated ST elevation during FPO. On the basis of theμCT analyses, significantly enlarged collateral arteries weremeasurable.

The treatment of the rats with ISDN retard (once daily intragastrally)also led to decreases in ST elevation during FPO, less arrhythmias andreduced infarct volumes. However, these improvements of infarctparameters are less distinct compared with NTG treatment. Moreover, theydid not show any significance.

Compared to controls, the treatment with ASA showed an impairment of ECGparameters and an increase of infarct volumes due to impaired collateralgrowth. These negative effects of ASA on arteriogenesis are alreadyknown. Interestingly, they can be partly abolished through an additionalNTG treatment (twice daily on buccal mucosa). Thus, collateral diameterswere enlarged in the ROI and ECG parameters were enhanced. Nevertheless,infarct volumes after FPO showed no reduction.

The SHAM groups did not differ among each other.

Further on, there were no differences measured between the Placebogroups and their corresponding control groups.

In conclusion, the presented results indicate that an intermittenttreatment with NTG solution decreases the size of an experimentallyinduced myocardial infarct.

In addition, effects on cardiac rhythm may ameliorate. These insightsare of outstanding relevance for clinical aspects.

Example 7

A preferred formulation prepared in accordance with the teachings hereinwill be tested as follows. Patients with a medical history of chronicstable exertional angina due to stable ischemic heart disease (coronaryartery disease) will receive a container of an aqueous GTN-containingpreparation. The container will be equipped with a pump spray devicewhich delivers a metered dose of 0.4 mg GTN per spray. Patients willsublingually self-administer the specified dose at the onset of anattack of angina pectoris. Patients will document the time lapsed fromtreatment to relief of the symptoms of angina pectoris, in particularanginal chest pain. It is expected that patients will experienceimmediate or near-immediate acute relief and return to a symptom-freecondition following treatment with the present invention.

Example 8

A preferred formulation prepared in accordance with the teachings hereinwill be tested as follows. In a placebo-controlled trial patients with amedical history of claudication intermittens due to peripheral arterydisease and a maximum walking distance of less than 250 m will receive acontainer of an aqueous GTN-containing preparation or placebo. Thecontainer will be equipped with a pump spray device which delivers ametered dose of 0.15 mg GTN per spray. Patients will sublinguallyself-administer the specified dose 5 minutes prior a daily walkingprogram of 45-60 minutes. Patients will be monitored at study inclusion(baseline measurement) as well as 8, 12 and 16 weeks after the start ofthe interventional phase to document their maximum and pain-free walkingdistances (by means of standard treadmill test) and their quality oflife (measured by a quality of life questionnaire) in comparison totheir baseline and placebo group. It is expected that patients treatedwith verum will show significant improvements over the placebo patients.GTN-receiving patients will show significantly improved walkingimpairment with a higher increase in maximum and pain-free walkingdistances and a higher quality of life due to GTN-induced higherincreased collateral circulation by means of arteriogenesis.

The invention claimed is:
 1. An aqueous pharmaceutical preparationcomprising: (a) a first component comprising 0.15 to 3 weight percent ofglyceryl trinitrate, (b) a second component comprising 40 to 95 weightpercent water, (c) a third component comprising 1 to 10 weight percentof at least one non-ionic water soluble polymer; and, (d) a fourthcomponent comprising 5 to 20 weight percent of ethanol; wherein thenon-ionic water soluble polymer is selected from the group of non-ionicpolymers consisting of: tyloxapol, poloxamer, and a mixture of tyloxapoland poloxamer.
 2. An aqueous pharmaceutical preparation comprising: (a)a first component comprising 0.15 to 3 weight percent of glyceryltrinitrate, (b) a second component comprising 40 to 95 weight percentwater, and; (c) a third component comprising 2 to 10 weight percent ofat least one non-ionic water soluble polymer, wherein the non-ionicwater soluble polymer is selected from the group of non-ionic polymersconsisting of: tyloxapol, poloxamer, and a mixture of tyloxapol andpoloxamer.
 3. The aqueous pharmaceutical preparation according to claim1 or 2 further comprising at least one additive selected from the groupconsisting of: a preservative, a taste component, a flavor component, asweetener, an acid, a base, and a buffering substance.
 4. The aqueouspharmaceutical preparation according to claim 3 wherein xylitol is ataste component.
 5. The aqueous pharmaceutical preparation according to3 wherein a buffering substance is present at about 0.1 to 1 weightpercent.
 6. The aqueous pharmaceutical preparation according to claim 1or 2 wherein the viscosity of the preparation is not more than 50 mPa*s,wherein viscosity is measured at 20 ° C. with a rotational viscosimeterat a shear rate of 1800/s.
 7. The aqueous pharmaceutical preparationaccording to claim 1 or 2 wherein the viscosity of the preparation isnot more than 40 mPa*s, wherein viscosity is measured at 20 ° C. with arotational viscosimeter at a shear rate of 1800/s.
 8. The aqueouspharmaceutical preparation according to claim 1 or 2 wherein theviscosity of the preparation is not more than 30 mPa*s, whereinviscosity is measured at 20 ° C. with a rotational viscosimeter at ashear rate of 1800/s.
 9. The aqueous pharmaceutical preparation of claim1 or 2 wherein the preparation is in the form of a spray.